Sulfamide as Zinc Binding Motif in Small Molecule Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

Article abstract of DOI:10.1021/acs.jmedchem.6b01276

Previously disclosed TAFIa inhibitors having a urea zinc-binding motif were used as the starting point for the development of a novel class of highly potent inhibitors having a sulfamide zinc-binding motif. High-resolution X-ray cocrystal structures were used to optimize the structures and reveal a highly unusual sulfamide configuration. A selected sulfamide was profiled in vitro and in vivo and displayed a promising ADMET profile.

Full text of DOI:10.1021/acs.jmedchem.6b01276

Brief Article
pubs.acs.org/jmc
Sulfamide as Zinc Binding Motif in Small Molecule Inhibitors of
Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)
Nis Halland,* Jorg Czech, Werngard Czechtizky, Andreas Evers, Markus Follmann,† Markus Kohlmann,
̈
Herman A. Schreuder, and Christopher Kallus*,‡
Sanofi R&D, Industriepark Hochst Building G838, D-65926 Frankfurt am Main, Germany
̈
S
* Supporting Information
ABSTRACT: Previously disclosed TAFIa inhibitors having a
urea zinc-binding motif were used as the starting point for the
development of a novel class of highly potent inhibitors having a
sulfamide zinc-binding motif. High-resolution X-ray cocrystal
structures were used to optimize the structures and reveal a
highly unusual sulfamide configuration. A selected sulfamide was
profiled in vitro and in vivo and displayed a promising ADMET
profile.
ntithrombotic agents usually act by preventing coagulation
by targeting proteases such as thrombin, factor Xa, or
We obtained an X-ray cocrystal structure of the complex. On
A
the basis of this and a report by Ryu et al.15 where a sulfamide
acts as zinc-binder in transition state analogue inhibitors of
carboxypeptidase A, we speculated that we could expand the
chemical space by replacing the urea zinc binder in our
previously reported TAFIa inhibitors with a sulfamide moiety.16
To explore this unusual zinc binding motif, we prepared the
direct sulfamide analogue of our previous lead structure (Figure
1) by the general synthesis route described in Scheme 1.
The key step in the synthesis of these analogues was
performed by first converting the side chain protected basic
amino acids to the corresponding N-sulfamidated oxazolidi-
nones 2 using chlorosulfonyl isocyanate at 0 °C in CH2Cl2
(Scheme 1). These oxazolidinones underwent subsequent
sulfamide formation by treatment with amino acid derivatives
4 in refluxing acetonitrile to afford the protected products 3.
Finally, the end products were obtained upon hydrogenolysis of
the benzyl ester and Cbz group using 10% Pd/C in methanol at
1 bar hydrogen pressure.
factor VIIa in the coagulation cascade or by addressing
antiplatelet targets like P2Y12 or GPVI.1 An alternative
approach would be to enhance fibrinolysis by targeting other
downstream enzymes such as factor XIIIa or TAFIa. As TAFIa
inhibition neither interferes with coagulation nor acts on
platelet function or influences other thrombin actions, it is
expected to present a novel antithrombotic mechanism with
obvious advantages such as a low associated risk of bleeding.
TAFI, a 423 amino acid protein, is synthesized in the liver
and present in plasma as a zymogen. When activated by
thrombin by proteolysis at Arg92, activated TAFIa cleaves
arginine and lysine residues on the surface of fibrin after initial
plasmin degradation and is thus a part of the fine-tuned
equilibrium of the coagulation−fibrinolysis system. By inhib-
ition of TAFIa, plasmin formation and endogenous fibrinolysis
are enhanced and an antithrombotic effect is achieved.2−7
TAFI, a member of the metallocarboxypeptidase family, has a
high selectivity for C-terminal basic amino acid side chains of
proteins and peptides.8−10 The cleavage of the C-terminal
peptide bond is catalyzed by coordination to a zinc ion present
in the active site.11
The direct sulfamide analogue of the highly potent urea lead
incorporating a (R)-configured cyclohexylalanine amino acid,
5a (Figure 1) turned out to be a less potent TAFIa inhibitor
with an IC50 of 75 μM, whereas the diastereoisomer 5b,
containing the (S)-configured cyclohexylalanine, showed a
significantly higher inhibitory activity with an IC50 of 0.33 μM.
In general, we found that the SAR of the sulfamide TAFI
inhibitors was very similar to that of the urea series when the
configuration of the nonbasic amino acid was changed from (R)
to (S). The reason for this was not obvious even when
RESULTS AND DISCUSSION
■
We have recently reported the optimization of a relatively large
anabaenopeptin natural product screening hit to a highly active
small molecule TAFIa inhibitor.12−14 A common feature of
these TAFIa inhibitors is the urea zinc binding motif where the
urea carbonyl binds directly to the zinc atom in the
metalloprotease. Early in our investigations we found the
moderately active sulfamide screening hit 1 with IC50 = 15 μM.
Received: August 26, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.6b01276
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Journal of Medicinal Chemistry
Brief Article
Figure 1. Sulfamide analogues 5a and 5b of the urea lead.
a
Scheme 1. Synthesis of Sulfamide TAFIa Inhibitors
a
Reagents and conditions: (a) chlorosulfonyl isocyanate, 2-bromoe-
thanol, triethylamine, CH2Cl2, 0 °C; (b) 1-hydroxybenzotriazole, 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N-
diisopropylethylamine, DMF, 20 °C; (c) TFA, CH2Cl2, 20 °C; (d)
triethylamine, MeCN, 80 °C; (e) H2 (1 bar), 10% Pd/C, MeOH, 20
°C.
Figure 2. Superposition of the crystal structures of tafCPB with the
urea inhibitor 9 (cyan; PDB code 4uib12) and the sulfamide inhibitor
7a (pink; PDB code 5lyi). (a) Overall view of the inhibitors. The
arrows in the insets show the N(1)−S(O2) and N(1)−C(O) bonds
with differing synclinal/antiperiplanar arrangements of the C* and
N(2) residues. The bonds are indicated in red in the corresponding
chemical structures. ap = antiperiplanar, sc = synclinal. (b) Close-up of
the interactions of the urea/sulfamide oxygens with the catalytic zinc.
considering the tetrahedral geometry of the sulfamide
compared to the planar geometry of the urea. Intrigued by
these results, we obtained X-ray cocrystal structures with
various ligands bound to a surrogate enzyme to compare the
two series. Due to the chemical instability of TAFIa, a
“TAFInized” carboxypeptidase B (tafCPB) was used (see ref 12
and Supporting Information S2). We thus obtained high-
resolution X-ray cocrystal structures of tafCPB complexed with
two related analogues, either urea 9 having an ((R)-configured
cyclohexyl-alanine moiety) or sulfamide 7a with an ((S)-
configured cyclohexylalanine moiety (Figure 2). The structures
showed that in both cocrystals the cyclohexyl rings and the
norbornyl groups are nearly superimposable. Likewise, the
amino and carboxy groups of the terminal lysine residues are
almost superimposable. In both structures the ligand and
enzyme are in the same configuration, and this is probably the
reason for the similar activity observed (7a IC50 = 12 nM; 9
IC50 = 3 nM). The opposite configuration at the chiral carbon
C* (adjacent to the sulfamide/urea moiety) was essential for
this highly similar arrangement: while 7a incorporated an (S)-
configured carbon C* and a synclinal (sc) arrangement of C*
and N(2) along the C*N(1)−S(O2)N(2) bond (indicated in
red, Figure 2), the (R)-configured C* and the antiperiplanar
(ap) arrangement of C* and N(2) along the C*N(1)−
C(O)N(2) bond led to a similar spatial arrangement in 9. A
search in the Cambridge Structural Database (CSD)17 for
small-molecule crystal structures with symmetrical urea and
sulfamide groups (excluding cyclic compounds) revealed a
sharp antiperiplanar distribution with an average torsion angle
of 173° for the urea compounds. In contrast, for sulfamides, a
broader synclinal distribution centered around 61° (see
Supporting Information S3) is found. No antiperiplanar torsion
angles were found for the sulfamides, indicating that the
antiperiplanar configuration along the second, adjacent SO2−
N(2) bond in Figure 2a must have been forced by the
B
DOI: 10.1021/acs.jmedchem.6b01276
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Journal of Medicinal Chemistry
Brief Article
complexation into the tafCBP active site, in line with the steep
SAR we observed. The cocrystal structures showed that the
catalytic zinc atom coordinated one of the two sulfamide
oxygen atoms in the outer shell at a distance of 3.3 Å
analogously to the coordination of the urea oxygen.
Interestingly, the other sulfamide oxygen was located at 2.0 Å
in the inner coordination shell of the catalytic zinc (Figure 2b).
The position of the zinc differs by 0.5 Å between the two
structures, with the zinc from the 9 complex shown in cyan and
the zinc from the 7a complex in gray. Interestingly, the oxygen
common between the urea and sulfamide complexes (circled) is
at 3.3 Å in the outer coordination shell of the zinc, while the
oxygen, unique to the sulfamide, is at 2.0 Å in the inner
coordination shell. On the basis of these results, we attempted
to improve the affinity. We prepared a small library, according
to Scheme 1, of sulfamides having an (S) configuration at the
nonbasic amino acid as in 5b. First, the 1-adamantyl or (R)-
(+)-bornyl amine groups of the terminal amide residue that
were previously identified as yielding highly active TAFI
inhibitors in the urea series were kept constant whereas the (S)-
cyclohexylalanine amino acid moiety was systematically
replaced by other (S)-configured amino acids with unfunction-
alized side chains (Table 1).
Replacement of the cyclohexylalanine amino acid by other
amino acids with very small or no R1 side chains, e.g., alanine 6f
or glycine 6e but also α-branched amino acid side chains such
as 6d or 6g, led to less active compounds. Generally, medium-
sized alkyl groups such as cyclopentylmethyl 7b, cyclo-
butylmethyl 7c, cyclopropylmethyl 7d, propyl 7h, or benzyl
7f afforded very high TAFIa affinity, but none of them were as
potent as the cyclohexylmethyl 7a side chain.
From the X-ray cocrystal structures of 9 and 7a it is observed
that cyclohexylmethyl side chain has an almost perfect fit into a
pocket in tafCPB rationalizing the high activity of 7a and 9. We
observed that the (R)-(+)-bornylamides were usually more
potent TAFIa inhibitors than the corresponding 1-adamanty-
lamides. Consequently, the combination of the most active (S)-
cyclohexylalanine amino acid with the (R)-(+)-bornylamide
resulted in the very potent inhibitor 7a with an IC50 of 12 nM.
Turning our attention to exploring (R)-(+)-bornyl and 1-
adamantyl replacements (R1), another small amide library was
generated (Table 2). Replacing the bridged bornyl or
adamantyl cycloalkanes by cyclohexyl or substituted cyclohexyl
derivatives led to compounds with reduced acctivity, whereas
the bicyclic amide 8f showed an improved IC50 of 0.67 μM
compared to the less active monocyclic and acyclic residues. A
number of additional amides incorporating bridged cyclo-
alkanes generally showed a very high activity on TAFIa as
exemplified by the (1S,2S,3S,5R)-(+)-isopinocampheylamide
8k with an IC50 of 0.13 μM.
Table 1. TAFIa Activity of Adamantylsulfamides 6 and (R)-
(+)-Bornylsulfamides 7: SAR of (S)-Cyclohexylalanine
Amino Acid Replacements
However, as in the urea series, the (R)-(+)-bornylamides still
delivered the most potent TAFIa inhibitors indicating a similar
binding mode. Interestingly, replacement of the (R)-(+)-bor-
nylamide by a (R)-(+)-bornyl ester as in 8m led to a more than
100-fold reduction in potency underlining the importance of
the amide geometry and the presence of a hydrogen bond
donor.
Keeping the (R)-(+)-bornylamide residue constant, we then
replaced the lysine side chain by other amino acid residues and
confirmed that a basic amino functionality is favorable for a
high binding activity (Table 3).
For instance, replacing the lysine amino acid with a nonbasic
phenylalanine as in 10c furnished a less potent inhibitor with
IC50 = 24 μM. On the other hand, not all basic residues led to
high binding affinity as exemplified by, for example, the N-
dimethyl substituted lysine 10a. Introduction of a nonbasic
hydrogen bond donor such as OH in compound 10b, an amide
residue as in 10d, or a urea as in 10f also led to nearly inactive
compounds. We therefore synthesized a number of sulfamides
where the lysine was replaced by other amino acids containing
less basic terminal groups such as aniline, imidazole, pyridine,
or 2-aminopyridine residues (10j−m). All these compounds
showed a good to excellent activity on TAFIa. Especially the 2-
aminopyridine 10m provided a highly active TAFI inhibitor
with an IC50 of 26 nM due to its ability make a bidentate
interaction with Asp255. As seen from Table 3 some basic
residues such as amidine (10p) or guanidine (10q) groups
provided highly active TAFIa inhibitors in the low nanomolar
range. Shortened linkers as in 10n where the C4 lysine side
chain was shortened to a C3 chain led to a reduction in activity
as compared to 7a. This is not surprising, since the natural
TAFI substrates contain lysine or arginine residues and their
respective C4 or C3 side chain is optimal for binding to the
enzyme. A similar effect was observed when comparing 10o and
10p.
C
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Journal of Medicinal Chemistry
Brief Article
Table 2. TAFIa Binding Affinity of Sulfamides: SAR of
Amide Substitution
Table 3. TAFIa Activity of (R)-(+)-Bornylsulfamides 10 with
Lysine Replacements
shown in Figure 3. Due to the high resolution (1.64−2.02 Å;
see Figure S1 and Table S1), the atomic positions are
accurately determined.
The coordinates of the inhibitors overlap almost perfectly,
indicating that their binding modes are nearly identical. The
only major difference observed, except for the differences due
to the presence of a urea or sulfamide linker, is that the
cyclohexyl group of the urea lead is rotated by 90° with respect
to the cyclohexyl group of the other inhibitors. Stacked parallel
to the aromatic ring of Tyr198, this orientation corresponds to
the lower energy conformation. While this conformation is
allowed by the compact norbornane group of 1, this
conformation is prohibited due to short contacts with the
more bulky bornane and adamantane groups in the other
inhibitors.
The hydrochloride salt of the potent sulfamide 7a was
selected for further in vitro eADMET and physicochemical
profiling to assess its potential as preclinical candidate (Table
4). 7a is a strong TAFI inhibitor with an IC50 of 12 nM, and
only a minor decrease in activity was observed in the presence
of 1% human serum albumin. The activity in human pooled
blood was also determined in a standard clot lysis assay and was
a
Mixture of diastereomers.
Besides the crystal structure of 7a-tafCPB complex, two other
high resolution crystal structures with the sulfamide ligands 1
and 6a were solved during the optimization. A superposition of
these complexes together with the urea 9 and the urea lead is
D
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Journal of Medicinal Chemistry
Brief Article
(Table 4). Furthermore, 7a, which was found to be stable when
incubated with human, mouse, or rat microsomes, showed a
very moderate intrinsic clearance in human hepatocytes and a
moderate CYP3A4 inhibition with IC50 = 12 μM at the
midazolam binding site. 7a did not display any CYP1A1,
CYP1A2, or CYP3A4 induction or hERG channel liability.
Lastly, 7a was tested against a panel of other proteases at 100
μM and none of them were inhibited significantly. Altogether,
7a displayed a very similar physicochemical and in vitro
ADMET profile compared the corresponding urea 9.12 Due to
the low Caco-2 permeability and high solubility, an intravenous
application was considered the preferred route of admin-
istration for in vivo studies.
Compound 7a was further profiled in an in vivo
pharmacokinetic study in rat to determine pharmacokinetic
parameters. After intravenous bolus administration of 1.0 mg/
kg, blood and urine samples were collected for up to 24 h and
the key pharmacokinetic parameters determined (Table 5). The
Figure 3. Superposition of the crystal structures of tafCPB with the
urea lead (green, PDB code 5lyf) and sulfamides 1 (magenta, PDB
code 5lyd), 7a (pink, 5lyi), 6a (orange, PDB code 5lyl), and 9 (cyan,
PDB code 4uib). The protein part (white) is from the highest
resolution structure with ligand 7a.
Table 5. Pharmacokinetic Parameters of 7a in Rat after
Intravenous Bolus Administration of 1 mg/kg
Table 4. Physicochemical and in Vitro ADMET Properties of
Sulfamide 7a
parameter
t1/2 (h)
1.7
C0 (ng/mL)
clearance (L h−1 kg−1
Vss (L/kg)
3800
0.70
0.38
4
)
% of dose excreted into urine after 24 h
compound is cleared relatively rapidly in rat with a terminal
half-life of 1.7 h, and 4% of the administered dose is excreted
into urine after 24 h. The volume of distribution at steady state
was calculated to be 0.38 L/kg with plasma concentrations
above IC50 for up to 4 h after administration but below
detectable levels after 24 h. Overall, this profile is compatible
with administration by iv infusion in the clinic as the relative
short half-life quickly leads to steady state plasma concen-
trations and enables a quick washout of the drug after treatment
has ended.
property
value
MW (parent)
514.73
0.457
2.02
4.52
159
solubility mg/mL (pH 7.4, 25 °C)
log D (pH 7.4, 25 °C)
ClogP
PSA (Å2)
H-bond donors
6
H-bond acceptors
9
rotatable bonds
12
LE (kcal/mol)/LLE
0.31/5.9
0.012
0.051
0.123
21
CONCLUSION
■
IC50 TAFIa (μM)
In summary, we have developed a novel class of small molecule
TAFIa inhibitors using a sulfamide moiety as an unusual zinc
binding motif. X-ray cocrystal structures elucidated the specific
interactions between ligand and enzyme. A series of sulfamides
exhibit a similar SAR compared to our previously reported urea
series when the configuration of one chiral center was reversed.
Further cocrystal structures were obtained to clarify this
unexpected result and revealed that this was due to a highly
unusual sulfamide configuration that allowed the same spatial
occupancy. The highly active sulfamide 7a displays an attractive
in vitro ADMET and in vivo PK profile suitable for further in
vivo efficacy studies.
IC50 TAFIa (μM) + 1% human serum albumin
IC50 clot lysis assay (μM)
metabolic degradation in human microsomes (%)
c
c
metabolic degradation in rat microsomes (%)
8
c
metabolic degradation in mouse microsomes (%)
0
intrinsic clearance in human hepatocytes ((mL/h)/106 cells)
Caco2 permeability (×10−7 cm/s)
0.043
0.6 (low)
12/>30
<10
a
CYP3A4 inhibition IC50 (μM) midazolam site/testosterone site
CYP1A1, CYP1A2, CYP3A4 induction (%)
b
hERG channel inhibition IC50, patch clamp (μM)
>10
other peptidases % inhibition at 100 μM FIIa, FVIIa, FXa, FXIa, <40
FXIIa, urokinase, C 1s, tPA, tryptase, trypsin, plasmin, KLKB1
d
a
b
Incubated at 37 °C for 10−30 min at 0.3−30 μM. Patch-clamp
EXPERIMENTAL SECTION
■
technique in the whole-cell configuration on recombinant chinese
hamster ovary (CHO) cells. % degradation after 20 min incubation
(see Supporting Information for details). Kallikrein B, plasma
c
X-ray Crystal Structure Determination. A mutated “tafinized”
porcine carboxypeptidase B (T111-L416; SwissProt sequence) was
used where 8 residues, closest to the active site that were different
between CPB and TAFI, were mutated to their TAFI equivalents.
These mutations were F175I, T302S, M309H, L311V, I355L, P357L,
A359P, and S362G. The recombinant protein was expressed in P.
pastoris GS115.The purified protein was dissolved in 50 mM Tris-HCl,
pH 7.5, and concentrated to 11 mg/mL. 1 μL of protein solution was
equilibrated against 1 μL of reservoir solutions containing 16−20%
d
(Fletcher factor) 1.
found to be high with IC50 = 0.123 μM. The high activity of 7a
was also reflected by a ligand efficacy (LE) of 0.31 kcal/mol and
high lipophilic ligand efficacy (LLE) of 5.9. 7a is relatively polar
with a log D of 2.0 and possesses a high aqueous solubility
E
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Brief Article
PEG3350, 100 mM MES, pH 5.5, and 50 mM Zn acetate. Crystals
were soaked with inhibitors by adding 1 μL of a 10 mM solution of
inhibitor in DMSO to a CPB crystal in 9 μL of reservoir solution. After
overnight incubation, the crystal was transferred to a drop of 8 μL of
soakbuffer with 2 μL of glycerol and the crystal was picked with a
nylon loop and flash frozen in liquid nitrogen. Data were collected at
the European Synchrotron Radiation Facility (ESRF). Data processing
and scaling were carried out using the XDS package. Model building
and inhibitor fitting was done with Quanta and Coot, and refinement
was done with Buster (1) and Refmac (urea lead and 7a and 8a). The
crystals diffracted to between 1.64 and 2.02 Å resolution with an
overall Rmeas between 7.1% and 11.0% (see Table S1). The resulting
maps were of excellent quality and clearly and unambiguously revealed
the binding modes of the inhbitors (see Figure S1).
[(S)-2-Cyclohexyl-1-((1R,2S,4R)-1,7,7-trimethylbicyclo[2.2.1]-
hept-2-ylcarbamoyl)ethyl]carbamic Acid tert-Butyl Ester (4a).
To a solution of 5.0 g of (S)-2-tert-butoxycarbonylamino-3-cyclohexyl-
propionic acid (Boc-Cha-OH, 18.4 mmol, 1.0 equiv) in DMF (60 mL)
under argon at 0 °C were added 3.53 g of 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (18.4 mmol, 1.0 equiv), 1.25
g of 1-hydroxybenzotriazole (9.2 mmol, 0.5 equiv), and 7.3 mL of
Hunig’s base, and the mixture was stirred for 30 min. Then 2.83 g of
̈
(R)-(+)-bornylamine (18.4 mmol, 1.0 equiv) and 3.7 mL of Hunig’s
̈
base were added and the reaction was stirred for 16 h at ambient
temperature. The reaction mixture was quenched with NaHCO3 (sat.,
aq) and extracted with EtOAc three times. The combined organic
phases were washed with water two times and dried over Na2SO4
before evaporation. Purification by flash chromatography using silica
gel as the stationary phase and heptane/EtOAc as the eluent afforded
6.58 g (88% yield) of the title compound as a colorless solid. 1H NMR
(DMSO-d6, 500 MHz) δ 0.65 (s, 3H), 0.80−0.91 (m, 3H), 0.82 (s,
3H) 0.89, (s, 3H), 1.06−1.41 (m, 7H), 1.37 (s, 9H), 1.56−1.74 (m,
8H), 2.06−2.16 (m, 1H), 4.02 (q, 2H, J = 7.6 Hz), 4.09 (m, 1H), 6.67
(d, 1H, J = 8.1 Hz), 7.56 (d, 1H, J = 8.6 Hz). MS (ES+) calcd: [M +
H] 407.33. Found: 407.32.
Chemistry. 1H NMR spectra were recorded in the indicated
deuterated solvent at 400 or 500 MHz. Purity of all compounds tested
in biological assays were determined to be >95% by LCMS.
All sulfamides were prepared following the procedure for ((S)-6-
amino-2-{[(S)-2-cyclohexyl-1-((1R,2S,4R)-1,7,7-trimethylbicyclo-
[2.2.1]hept-2-ylcarbamoyl)ethylsulfamidyl]}hexanoic acid 7a
(S)-6-Benzyloxycarbonylamino-2(2-oxo-oxazolidine-
sulfonylamino)hexanoic Acid Benzyl Ester (2). To a solution of
5.21 g of chlorosulfonyl isocyanate (36.9 mmol, 1.0 equiv) in
dichloromethane (300 mL) under argon at 0 °C was slowly added a
solution of 2.61 mL of 2-bromoethanol (36.9 mmol, 1.0 equiv) in
dichloromethane (20 mL) at a rate so that the internal temperature
was kept below 10 °C. After addition the reaction mixture was allowed
to stir for an additional 30 min at 0 °C. To this solution was dropwise
added a mixture of 15.00 g of H-Lys(Z)-OBzL·HCl (36.9 mmol, 1.0
equiv) and 16.5 mL of triethylamine (118.0 mmol, 3.2 equiv) in 120
mL of CH2Cl2, keeping the temperature of the reaction mixture below
10 °C. After addition the ice bath was removed and the mixture stirred
at ambient temperature for 4 h. Then the reaction mixture was washed
three times with 100 mL of 0.2 M HCl (aq), dried over Na2SO4, and
evaporated to afford 18.4 g of the crude title compound 2 as a colorless
(S)-6-Amino-2-{[(S)-2-cyclohexyl-1-((1R,2S,4R)-1,7,7-
trimethylbicyclo[2.2.1]hept-2-ylcarbamoyl)ethylsulfamidyl]}-
hexanoic Acid (7a). 9.0 g of (S)-6-benzyloxycarbonylamino-2-{[(S)-
2-cyclohexyl-1-((1R,2S,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-
ylcarbamoyl)ethylsulfamidyl]}hexanoic acid benzyl ester 3a (12.2
mmol) was dissolved in 90 mL of methanol, 600 mg of 10% Pd/C was
added, and the reaction flask was kept under a hydrogen atmosphere at
ambient temperature and pressure and stirred for 3.5 h. The reaction
mixture was then filtered using Celite filter aid and evaporated under
vacuum to afford 6.1 g (97%) of the title compound as a colorless oil.
The product was dissolved in MeCN and water was added to afford a
suspension in a 1:10 MeCN/water mixture. The solvents were
removed by freeze-drying to afford the title compound as a colorless
1
solid. H NMR (DMSO-d6, 500 MHz) δ 0.68 (s, 3H), 0.82 (s, 3H),
0.83−0.91 (m, 2H), 0.89 (s, 3H), 0.97 (dd, 1H, J = 4.8, 13.0 Hz),
1.08−1.34 (m, 7H), 1.35−1.55 (m, 5H), 1.56−1.72 (m, 9H), 1.78 (d,
1H, J = 13.0 Hz), 2.04−2.13 (m. 1H), 2.75 (t, 2H, J = 7.1 Hz), 3.51 (t,
1H, J = 5.5 Hz), 3.83 (t, 1H, J = 7.0 Hz), 4.03−4.10 (m, 1H), 6.91−
7.05 (br, 1H), 7.77 (d, 1H, J = 8.8 Hz), 7.5−8.2 (br, 2H). MS (ES+)
calcd: [M + H] 515.33. Found: 515.35.
1
oil that was used directly in the next step. H NMR (DMSO-d6, 500
MHz) δ 1.24−1.44 (m, 4H), 1.54−1.63 (m, 1H), 1.68−1.77 (m, 1H),
2.95 (q, 2H, J = 6.5 Hz), 3.85 (q, 1H, J = 7.8 Hz), 3.94 (q, 1H, J = 8.6
Hz), 4.08−4.14 (m, 1H), 4.27 (t, 2H, J = 8.2 Hz), 5.00 (s, 2H), 5.12
(s, 2H), 7.18−7.25 (m, 1H), 7.27−7.42 (m, 10H), 9.05 (d, 1H, J = 8.4
Hz). MS (ES+) calcd: [M + H] 520.17. Found: 520.30.
(S)-6-Benzyloxycarbonylamino-2-{[(S)-2-cyclohexyl-1-
((1R,2S,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-ylcarbamoyl)-
ethylsulfamidyl]}hexanoic Acid Benzyl Ester (3a). To a solution
of 6.5 g of [(S)-2-cyclohexyl-1-((1R,2S,4R)-1,7,7-trimethylbicyclo-
[2.2.1]hept-2-ylcarbamoyl)ethyl]carbamic acid tert-butyl ester 4a
(16.0 mmol) in 50 mL of CH2Cl2 under argon at 0 °C was slowly
added 50 mL of TFA, and the reaction was allowed to warm to
ambient temperature. After 3 h the reaction mixture was evaporated to
remove excess TFA and CH2Cl2 to afford 4.9 g of crude (S)-2-amino-
3-cyclohexyl-N-((1R,2S,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)-
propionamide trifluoroacetic acid salt (16.0 mmol, 1.0 equiv) as a
slightly yellow oil that was dissolved in MeCN (80 mL), and 8.9 mL of
Et3N was added together with 11.63 g of (S)-6-benzyloxy-
carbonylamino-2(2-oxo-oxazolidinesulfonylamino)hexanoic acid ben-
zyl ester 2 (22.4 mmol, 1.4 equiv). The reaction mixture was heated to
reflux for 20 h. After cooling, the volatiles were evaporated and the
crude reaction mixture was purified directly by flash chromatography
using silica gel as the stationary phase and heptane/EtOAc as the
eluent. This afforded 9.0 g (76% yield) of (S)-6-benzyloxy-
carbonylamino-2-{[(S)-2-cyclohexyl-1-((1R,2S,4R)-1,7,7-trimethyl-
bicyclo[2.2.1]hept-2-ylcarbamoyl)ethylsulfamidyl]}hexanoic acid ben-
zyl ester 3a as a colorless foam after evaporation. 1H NMR (DMSO-d6,
500 MHz) δ 0.68 (s, 3H), 0.79−0.92 (m, 3H), 0.81 (s, 3H) 0.86, (s,
3H), 1.05−1.43 (m, 12H), 1.37, 1.53−1.79 (m, 10H), 2.06−2.15 (m,
1H), 2.93 (q, 2H, J = 6.4 Hz), 3.73−3.83 (m, 2H), 4.08 (m, 1H), 4.99
(s, 2H), 5.12 (d, 2H, J = 5.2 Hz), 6.96 (d, 1H, J = 8.9 Hz), 7.18−7.22
(m, 2H), 7.28−7.41 (m, 10H), 7.64 (d, 1H, J = 8.8 Hz). MS (ES+)
calcd: [M + H] 739.41. Found: 739.43.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01276.
Representative experimental procedures for X-ray
crystallography, synthesis, biochemical assays, and
analytical data for all compounds (PDF)
Molecular formula strings and some data (CSV)
Accession Codes
Atomic coordinates and structure factors for the urea lead and
compounds 1, 6a, and 7a can be accessed using PDB codes
5lyf, 5lyd, 5lyl, and 5lyi, respectively. The authors will release
the atomic coordinates and experimental data upon article
publication.
AUTHOR INFORMATION
■
Corresponding Authors
*N.H.: phone, +49-69305-36193; e-mail, Nis.Halland@sanofi.
com.
*C.K.: phone, +49-69305-34322; e-mail, christopher.kallus@
bayer.com.
F
DOI: 10.1021/acs.jmedchem.6b01276
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
(15) Park, J. D.; Kim, D. H.; Kim, S.-J.; Woo, J.-R.; Ryu, S. E.
Sulfamide-based inhibitors for carboxypeptidase A. Novel type
transition state analogue inhibitors for zinc proteases. J. Med. Chem.
2002, 45, 5295−5302.
(16) Akparov, V.; Sokolenko, N.; Timofeev, V.; Kuranova, I.
Structure of the complex of carboxypeptidase B and N-sulfamoyl-L-
arginine. Acta Crystallogr., Sect. F: Struct. Biol. Commun. 2015, 71,
1335−1340.
Present Addresses
†M.F.: Bayer Pharma AG, Aprather Weg 18A, D-42113
Wuppertal, Germany.
‡C.K.: Bayer Cropscience, Industriepark Hochst Building G836,
D-65926 Frankfurt am Main, Germany.
̈
Notes
The authors declare no competing financial interest.
(17) Allen, F. H. The Cambridge Structural Database: a quarter of a
million crystal structures and rising. Acta Crystallogr., Sect. B: Struct. Sci.
2002, 58, 380−388.
ACKNOWLEDGMENTS
■
The authors thank Stefan Dierl and Britta Bohnisch for the
̈
preparation of tafinized CPB, and Alexander Liesum and Petra
Loenze for growing the crystals and help with data processing
and refinement.
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J. Med. Chem. XXXX, XXX, XXX−XXX