Active site mapping of trypsin, thrombin and matriptase-2 by sulfamoyl benzamidines

DOI: 10.1016/j.bmc.2012.08.042

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Bioorganic and medicinal chemistry p. 6489 - 6505,17 (2012)

Update date:2022-07-30

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Authors:

Dosa, Stefan

Stirnberg, Marit

Luelsdorff, Verena

Haeussler, Daniela

Maurer, Eva

Guetschow, Michael

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Article abstract of DOI:10.1016/j.bmc.2012.08.042

The benzamidine moiety, a well-known arginine mimetic, has been introduced in a variety of ligands, including peptidomimetic inhibitors of trypsin-like serine proteases. According to their primary substrate specificity, the benzamidine residue interacts with the negatively charged aspartate at the bottom of the S1 pocket of such enzymes. Six series of benzamidine derivatives (1-73) were synthesized and evaluated as inhibitors of two prototype serine proteases, that is, bovine trypsin and human thrombin. As a further target, human matriptase-2, a recently discovered type II transmembrane serine protease, was investigated. Matriptase-2 represents an important regulatory protease in iron homeostasis by down-regulation of the hepcidin expression. Compounds 1-73 were designed to contain a fixed sulfamoyl benzamidine moiety as arginine mimetic and a linker-connected additional substructure, such as a tert-butyl ester, carboxylate or second benzamidine functionality. A systematic mapping approach was performed with these inhibitors to scan the active site of the three target proteases. In particular, bisbenzamidines, able to interact with both the S1 and S3/S4 binding sites, showed notable affinity. In branched bisbenzamidines 66-73 containing a third hydrophobic residue, opposite effects of the stereochemistry on trypsin and thrombin inhibition were observed.

Full text of DOI:10.1016/j.bmc.2012.08.042

Bioorganic & Medicinal Chemistry 20 (2012) 6489–6505

Contents lists available at SciVerse ScienceDirect

Bioorganic & Medicinal Chemistry

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Active site mapping of trypsin, thrombin and matriptase-2 by sulfamoyl

benzamidines

Stefan Dosa ^a, Marit Stirnberg ^a, Verena Lülsdorff ^a, Daniela Häußler ^a,b, Eva Maurer ^a,

Michael Gütschow ^a,b,

⇑

^aPharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany

^bNRW International Graduate Research School Biotech-Pharma, Bonn, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:

The benzamidine moiety, a well-known arginine mimetic, has been introduced in a variety of ligands,

including peptidomimetic inhibitors of trypsin-like serine proteases. According to their primary substrate

speciﬁcity, the benzamidine residue interacts with the negatively charged aspartate at the bottom of the

S1 pocket of such enzymes. Six series of benzamidine derivatives (1–73) were synthesized and evaluated

as inhibitors of two prototype serine proteases, that is, bovine trypsin and human thrombin. As a further

target, human matriptase-2, a recently discovered type II transmembrane serine protease, was investi-

gated. Matriptase-2 represents an important regulatory protease in iron homeostasis by down-regulation

of the hepcidin expression. Compounds 1–73 were designed to contain a ﬁxed sulfamoyl benzamidine

moiety as arginine mimetic and a linker-connected additional substructure, such as a tert-butyl ester, car-

boxylate or second benzamidine functionality. A systematic mapping approach was performed with

these inhibitors to scan the active site of the three target proteases. In particular, bisbenzamidines, able

to interact with both the S1 and S3/S4 binding sites, showed notable afﬁnity. In branched bisbenzami-

dines 66–73 containing a third hydrophobic residue, opposite effects of the stereochemistry on trypsin

and thrombin inhibition were observed.

Received 10 July 2012

Accepted 16 August 2012

Available online 5 September 2012

Keywords:

Active site mapping

Benzamidines

Bisbenzamidines

Matriptase-2

Serine proteases

1. Introduction

O-acetylamidoximes,^19–23a protocol that was utilized in the course

of this study.

The benzamidine moiety is an important pharmacophore in

medicinal chemistry prone to interact with protein regions bearing

anionic and hydrogen bond acceptor groups. To improve their oral

bioavailability, amidoximes have been successfully introduced as

prodrugs which are metabolized to the active benzamidines

in vivo by cytochrome P450 or other NAD(P)H-dependent

oxidoreductases.¹

Several synthetic routes to benzamidines have been reported,

such as the conversion of aromatic imidates (Pinner reaction)^2–6

or thioimidates^5,7–11by amination. The reaction of benzonitriles

with aluminum amides, prepared from trimethylaluminum,^12,13

or lithium bis(trimethylsilyl)amide also proved to furnish benz-

amidines.^14–16Furthermore, they are accessible via catalytic reduc-

tion of amidoximes, obtained from nitriles, by taking advantage of

potassium or ammonium formate as hydrogen source.^13,17This

method was been applied to the synthesis on solid-phase support

by reducing polymer-bound amidoximes using SnCl₂Â 2H₂O.¹⁸

Benzamidines can also be obtained by catalytic hydrogenation of

The bisbenzamidine scaffold exhibits a broad spectrum of anti-

protozoal activity, mainly directed against the parasites Plasmo-

dium falciparum, Trypanosoma brucei, and Leishmania donovani,

which cause considerable morbidity and mortality,^24,25as well as

antifungal activity beneﬁcial for the treatment of AIDS-related

Pneumocystis carinii pneumonia.^25,26

Benzamidines have been shown to act as excellent arginine side

chain mimetics²⁷to address targets where arginine is involved as a

part of their natural ligand binding sequence. For example, benz-

amidines based upon the Arg-Gly-Asp sequence of the

a-chain of

ﬁbrinogen are antagonists of the platelet membrane glycoproteins

IIb/IIIa, that is, the ﬁbrinogen receptors.^8,28Benzamidine contain-

ing imidazolyl derivatives act as antagonists of the presynaptic his-

tamine H₃receptor.¹³

Because of the arginine mimicking properties, benzamidine

substructures have been extensively utilized in the rational design

of inhibitors for trypsin-like serine proteases which are character-

ized by their primary substrate speciﬁcity for arginine in P1 posi-

tion. Examples include inhibitors for the target enzymes trypsin,⁹

thrombin,^4,5factor Xa,^{6,10,11,16,21,29,30}urokinase,²²plasmin,³¹and

matriptase-1.²³Among them, the thrombin inhibitors melagatran

and dabigatran were introduced into the market, in form of their

⇑

Corresponding author.

E-mail address: guetschow@uni-bonn.de (M. Gütschow).

http://dx.doi.org/10.1016/j.bmc.2012.08.042

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S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

double prodrugs.^32,33A binding mode for benzamidine inhibitors

and trypsin-like serine proteases has been experimentally con-

ﬁrmed and is generally accepted, inasmuch as the benzamidine

group is deeply buried in the S1 speciﬁcity pocket where a nega-

tively charged conserved Asp189 at the bottom is engaged in a salt

bridge with the positively charged benzamidine functionality.^34,35

The architecture of the S1 pocket of trypsin and thrombin resem-

bles that of matriptase-2, a recently discovered serine protease.

As trypsin and thrombin, matriptase-2 has a primary substrate

speciﬁcity for basic amino acids in P1 position.^36–39A homology

model of matriptase-2 was generated showing that binding of argi-

nine to the S1 site is likewise stabilized by a salt bridge to the spec-

iﬁcity determining residue Asp756 (chymotrypsinogen numbering

189).³⁹

benzamidines as well as the introduction of the amide bioisosteric

sulfonamide moiety.⁴⁵Throughout the various series of products,

the meta- or para-substitution pattern was incorporated into the

benzamidine products as a ﬁrst point of diversity.

Sulfonamides resulting from aromatic amines and cya-

nobenzenesulfonyl chlorides were found to react with a second

sulfonyl chloride in the presence of DIPEA to produce undesired

bis-sulfonylated amines. Therefore, pyridine, as a weaker base,

was used to synthesize sulfonamides B (Table 1). Compounds B

were reacted with hydroxylamine, and the intermediate amidox-

imes were directly acetylated, followed by palladium-catalyzed

hydrogenolysis. The process was optimized in terms of repressing

the formation of 1,2,4-oxadiazole by-products.⁴⁶Benzamidines 1–

16 were efﬁciently precipitated either as acetate salts or as dipolar

ions in excellent purity by addition of deep cooled, dry ethyl ace-

tate to the oily residue which was obtained after ﬁltration and

evaporation of the solvent of the reduction step. Finally, lyophiliza-

tion from aqueous HCl furnished the benzamidine hydrochlorides.

With this powerful method of isolation in hand, we could use it for

the other types of benzamidines (Tables 2–6) and adapt it to their

physico-chemical properties. The majority of these products, that

is, 5–16, inhibited bovine trypsin with K_ivalues between 16 and

50 lM. The binding afﬁnities of 1–16 to thrombin were weak with

a slight preference for the meta-substituted benzamidines. Matrip-

tase-2 was not affected by these small benzamidines, except by

compound 1 exhibiting a K_ivalue of 88 lM.

To further map the active sites of the three proteases, we de-

signed molecules which still contain the sulfamoyl benzamidine

part connected via linkers of various lengths to a dimethoxyben-

zene moiety. As this hydrophobic substructure was maintained,

the following series (Table 2) might allow to estimate the inﬂuence

of the linker structure on the biological activity. Amine hydrochlo-

Matriptase-2 belongs to the type II transmembrane serine pro-

teases (TTSPs). One member of this family of cell surface proteases,

matriptase-1, possesses an extracellular matrix degrading activity

and represents a potential therapeutic target for cancer ther-

apy.^23,40The closely related enzyme matriptase-2, encoded by

the gene TMPRSS6, is predominantly expressed in the liver and con-

sists of a short N-terminal cytoplasmic tail, a single transmem-

brane domain, and a composite ectodomain with a C-terminal

serine protease domain.^36,41In vitro, matriptase-2 can degrade

extracellular matrix components such as ﬁbronectin, ﬁbrinogen

and type I collagen, but with lower efﬁciency than matriptase-

1.³⁶However, enhanced expression of matriptase-2 signiﬁcantly

reduced the invasive breast and prostate cancer growth, and de-

creased levels of matriptase-2 correlate with poor patient outcome,

suggesting that matriptase-2 participates in regulating cellular

migration.⁴²The recently discovered role of matriptase-2 in iron

homoeostasis has attracted much attention. Mutations in TMPRSS6

lead to a loss of matriptase-2 proteolytic activity and are associated

with iron-refractory iron-deﬁciency anemia (IRIDA) due to ele-

vated levels of the liver hormone hepcidin.⁴³Matriptase-2 inhibits

hepcidin synthesis at the transcriptional level through proteolytic

cleavage of the co-receptor hemojuvelin suppressing SMAD signal-

ing which results in increased plasma iron levels.⁴⁴A cell mem-

brane localization of an inactive zymogen has been demonstrated

using HEK-293 cells, stably transfected with cDNA encoding hu-

man matriptase-2.³⁸Matriptase-2 undergoes shedding via a trans

mechanism and accumulates in the conditioned medium as an

activated form containing the catalytic domain. The conditioned

medium was used as a source for matriptase-2 activity to deter-

mine K_ivalues of the ﬁrst matriptase-2 inhibitors.³⁹Matriptase-2

inhibitors could be beneﬁcial as pharmacological tools to further

investigate its exact role in regulating iron homeostasis and might

also be useful for therapeutic intervention of frequent iron disor-

ders such as systemic iron overload (hemochromatosis) or iron

loading anemias where the level of hepcidin is inappropriately low.

Herein, we present a combinatorial approach to map the active

site of trypsin-like serine proteases with polyfunctionalized benz-

amidines. The compounds were designed to contain a ﬁxed sulfa-

moyl benzamidine moiety for accommodation in the S1 pocket

and several linker-connected functionalities for additional interac-

tions with the S3/S4 binding region. We have prepared six series of

benzamidine derivatives and evaluated these compounds towards

three selected serine proteases, including matriptase-2, in ﬂuoro-

metric enzyme assays.

rides D were obtained from Boc-protected glycine, b-alanine and c-

aminobutyric acid C, respectively, which were coupled with 3,4-

dimethoxyaniline or the more ﬂexible 3,4-dimethoxybenzylamine

using the mixed anhydride method and deprotected with gaseous

hydrogen chloride. Pyridine had to be replaced by DIPEA for the

formation of the sulfonamides E, which were converted to benz-

amidines according to a slightly modiﬁed protocol. Thereby, the

oily residues, obtained after hydrogenolysis, were dissolved in ace-

tic acid and rapidly injected into deep cooled, dry ethyl acetate.

Gaseous hydrogen chloride was passed through the resulting solu-

tion to precipitate the benzamidine hydrochlorides 17–28. Initial

attempts to start the sequence with the unprotected amino acid

and the sulfonyl chloride, followed by mixed anhydride coupling

with the dimethoxy-substituted amine led to an additional car-

bamoylation at the sulfonamide nitrogen.

The inhibitory activity of benzamidines 17–28 towards trypsin

was similar compared to that of 1–16. However, the insertion of

a linker led to a stronger affection of thrombin, in particular in

meta-substituted compounds (12 and 16 vs 23–28). All of these

meta derivatives were more active than their para-substituted iso-

mers 17–22. The same trends were observed for matriptase-2,

where only two meta compounds, 26 and 28 with the extended

c-amino butyric acid-based linker (n = 3), were inactive.

To verify the account of the hydrophobic residue at the site

opposite to the benzamidine, an additional set of compounds

was synthesized (Table 3). Glycine derivatives 29–32 contain var-

ious groups in place of the dimethoxyphenyl moiety of 23, which

exhibited the lowest K_ivalue of the previous series. By using aque-

ous triﬂuoroacetic acid, the ﬁnal compounds 29–32 were obtained

as triﬂuoroacetates after lyophilization. The replacement of the

3,4-dimethoxyphenyl by the less electron-rich phenyl (in 29) or

2. Results and discussion

Our initial results towards the synthesis of benzamidines indi-

cated the conversion of nitriles to be the most practical synthetic

route.¹⁹Thus, we considered 3- and 4-cyanobenzenesulfonyl

chloride to be attractive building blocks for the formation of

the 2-naphthyl ring with an extended

nonaromatic cyclohexyl ring (in 31) resulted in similar activity

proﬁles. Thus, interactions seem not to be essential for a

p-system (in 30) or the

p–p

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

6491

Table 1

Synthesis and biological evaluation of benzamidines 1–16

O₂

3- or 4-cyanobenzenesulfonyl

R¹

NH₂chloride, pyridine

R²

1. H₂NOH×HCl, DIPEA

2. Ac₂O

3. H₂, Pd/C

4. HCl (0.1 N), lyophilization

O₂

R¹

R²

NH₂

1-16

Compd

R¹

R²

Arom. subst.

Bovine trypsin K_i^a

(lM)

Human thrombin K_i^a

(

lM)

Human matriptase-2 K_i^a

(lM)

OMe

para

meta

n.i.

230 23

n.i.

223 20

n.i.

135

n.i.

150

n.i.

OMe

160 18

165

120

102

245 20

165 11

n.i.

OMe

n.i.: no inhibition relates to IC₅₀values of >500

lM. IC₅₀values were determined from duplicate measurements with ﬁve different inhibitor concentrations. The substrate

concentration of 40

K_i= IC₅₀/(1+[S]/K_m).

lM refers to 1.81 Â K_m(bovine trypsin), 1.01 Â K_m(human thrombin) and 1.24 Â K_m(matriptase-2). K_ivalues were calculated using the equation

proposed accommodation in the S3/S4 binding pocket of the prote-

ases. The presence of the less bulky aliphatic isopropyl residue (in

32) reduced the inhibitory activity against matriptase-2. A compar-

ison of the amide linker-connected compounds 29 (Table 3) and 23

(Table 2) with their analogues lacking the amide group (13 and 16,

Table 1) revealed an advantageous effect of the additional CONH

unit present in 29 and 23.

Next, we focused on a further modiﬁcation of the linker struc-

ture connecting the meta benzamidine core with the hydrophobic

moiety. As depicted in Table 4, Boc-glycine was reacted with

Table 2

Synthesis and biological evaluation of benzamidines 17–28 with dimethoxybenzene substructure

3- or 4-cyanobenzene-

sulfonyl chloride, DIPEA

1. NMM, ClCO -Bu, amine

₂i

2. HCl

MeO

NH₃

NHBoc

1. H₂NOH×HCl, DIPEA

2. Ac₂O

NH₂

3. H₂, Pd/C

4. HCl (0.1 N),

5. lyophilization

MeO

O₂

17-28

Compd

Arom. Subst.

Bovine trypsin K_i^a

(lM)

Human thrombin K_i^a

(

Human matriptase-2 K_i^a

(lM)

para

meta

11.4 0.1

13.6 0.6

250 26

187 11

115 17

n.i.

172

n.i.

20.2 0.6

26.9 0.8

175

n.i.

6.4 0.4

14.2 0.2

4.8 0.3

15.6 0.6

12.3 0.4

82 10

132 16

155 24

n.i.

187 30

n.i.

15.6 0.9

For assay details, see Table 1.

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S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

Table 3

Synthesis and biological evaluation of benzamidines 29–32 with modiﬁed hydrophobic substructures

3-cyanobenzenesulfonyl

chloride, DIPEA

1. NMM, ClCO -Bu, amine

₂i

2. H₂, Pd/C

NH₂

NHCbz

1. H₂NOH×HCl, DIPEA

2. Ac₂O

3. H₂, Pd/C

4. TFA (0.1 N), lyophilization

NH₂

F₃CCO₂

O₂

NH₂

29-32

Human thrombin K_i^a

4.0 0.2

7.6 0.8

Compd

Bovine trypsin K_i^a

(

lM)

Human matriptase-2 K_i^a

(lM)

10.9 0.7

5.58 0.09

12.6 0.5

2-Naphthyl

i-Pr

148

For assay details, see Table 1.

3-aminobenzonitrile and deprotected to the ammonium chloride H

which was then converted with various sulfonyl chlorides to ni-

triles I. Following the protocol already applied for the previous four

benzamidines, the products 33–35 were synthesized. Among them,

33 and 34 share the substitution pattern of benzamidines 23 (Table

2) and 30 (Table 3), respectively, but possess the inverted pepti-

domimetic chain. Actually, this modiﬁcation diminished the afﬁn-

ity for trypsin and matriptase-2 (33 vs 23; 34 vs 30). For the design

of further compounds, we concluded to maintain the non-inverted

linker structure and thus to continue using 3- and 4-cya-

nobenzenesulfonyl chloride as precursors for the P1 position.

At this point of the study, we decided to systematically map the

binding area of the S3/S4 pocket of the selected serine proteases by

introducing a tert-butoxycarbonyl or a carboxy residue into the

aromatic ring at the site opposite to the benzamidine. For this pur-

pose, a complete combination was achieved with respect to three

points of diversity, (i) the position of the tert-butoxycarbonyl and

carboxy residues (3- or 4-position), (ii) the length of the linker

(n = 1–3), and (iii) the orientation of the amidine group (para or

meta). The corresponding ﬁnal products, tert-butyl benzoates 36–

47 and benzoic acid derivatives 48–59 are displayed in Table 5.

Coupling of the Cbz-protected amino acids J with tert-butyl 4-ami-

nobenzoate or tert-butyl 3-aminobenzoate and deprotection affor-

ded intermediates K. The required aniline derivatives were

obtained from the respective nitrobenzoic acids upon esteriﬁcation

with oxalyl chloride and tert-butanol followed by reduction of the

nitro group. The transformation into sulfamoyl benzamidines 36–

47 was carried out via cyano-substituted sulfonamides L. The

tert-butyl ester group was not affected under the conditions used

for the preparation of 36–47. The second group of test compounds,

benzoic acid derivatives 48–59, was prepared by triﬂuoroacetic

acid-promoted cleavage of tert-butyl esters 36–47. Overall, the

route to 36–47 and 48–59 included eight or nine synthetic steps,

respectively.

para-substituted ones. Consequently, the benzoic acid derivatives

with a para-benzamidine substructure (48–53) are mainly inactive,

whereas the tert-butyl benzoates with a meta benzamidine sub-

structure (42–47) represent the most active inhibitors of this ser-

ies. For matriptase-2 inhibition, a tert-butoxycarbonyl/carboxy

substitution at position 4 was favored over position 3. Thus, the

meta benzamidine 44 containing the b-alanine linker and the

tert-butyl ester group at 4-position (K_i= 7.6 lM) was identiﬁed as

a substance inhibiting matriptase-2 in the lower micromolar range

with some selectivity over thrombin. Noticeably, the best matrip-

tase-2 inhibitor among the benzoic acid derivatives, 56

(K_i= 52

group of 12 meta benzamidines, only 47 and 59 with an incorpo-

rated -amino butyric acid (n = 3) did not inhibit matriptase-2.

lM), shares the substitution pattern of 44. Within the

This ﬁnding reinforces the preference of matriptase-2 for shorter

linker lengths (see Table 2, compounds 26 and 28).

It could be concluded that the negatively charged carboxylic

group did not contribute to attractive interactions between ben-

zoic acid derivatives and the S3/S4 binding site of thrombin and

matriptase-2. To investigate the outcome of positively charged

substructures, we introduced a second benzamidine functionality,

for which 4-cyanobenzylamine was used as precursor (Table 6).

Besides glycine, b-alanine, and

-aminobutyric acid (R¹= R²= H,

n = 1–3), both enantiomers of phenylalanine and O-benzylserine

(n = 1) were selected to function as linker structures. Ammonium

salts N were obtained via mixed anhydride method from Boc-pro-

tected amino acids C or M, respectively, and 4-cyanobenzylamine,

followed by deprotection. Dinitriles O were obtained from N and 3-

or 4-cyanobenzenesulfonyl chloride and were subsequently trans-

formed to bisbenzamidines 60–73 using the standard protocol for

the conversion of benzonitriles to benzamidines. The ﬁnal products

60–73 were afforded after puriﬁcation using a reversed phase

HPLC.

The biological data of the unbranched bisbenzamidines, that is,

60–65, were initially compared with the analogous dimethoxyben-

zyl derivatives (17–28, m = 1; Table 2). Such a comparison allowed

to assess the effect of an exchange of the dimethoxybenzene ring

by the second benzamidine moiety for the interaction with the

proposed S3/S4 binding pocket. In nearly all of the 18 cases, com-

pounds 60–65 were more active than their dimethoxy counter-

parts at trypsin, thrombin, and matriptase-2, respectively.

Examples for a pronounced improvement of the afﬁnity include

In general, trypsin was inhibited by these compounds to a sim-

ilar extent as observed for the other benzamidines noted above.

Rather minor differences in the trypsin-inhibiting activity of 36–

59 were determined, irrespective of the presence of the ester or

acid group, the linker structure and the positions of the aromatic

substituents. Comparable trends in the structure-activity relation-

ships of 36–59 were found both for thrombin and matriptase-2, as

follows. The tert-butyl esters 36–47 showed an enhanced activity

compared with the benzoic acid derivatives 48–59. The afﬁnities

of the meta-substituted benzamidines exceeded those of the

trypsin inhibition by 61 (K_i= 0.53 vs 26.9

lM of 21), thrombin inhi-

bition by 65 (K_i= 5.1 vs 60 M of 28), and matriptase 2 inhibition

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

6493

Table 4

Synthesis and biological evaluation of benzamidines 33–35 with an inverted chain

1. NMM, ClCO₂i-Bu,

2. 3-aminobenzonitrile

2. HCl

sulfonyl chloride, DIPEA

BocHN

H₃N

1. H₂NOH×HCl, DIPEA

2. Ac₂O

3. H₂, Pd/C

4. TFA (0.1 N), lyophilization

NH₂

O₂

NH₂

F₃CCO₂

Human matriptase-2 K_i^a

(lM)

33-35

Compd

Bovine trypsin K_i^a

29.4 0.9

19.2 0.9

Human thrombin K_i^a

3,4-(OMe)₂Ph

2-Naphthyl

1-Naphthyl

10.6 0.6

8.53 0.41

5.82 0.37

196

163

For assay details, see Table 1.

by 64 (K_i= 8.9 vs 187

M of 27). Introduction of b-alanine or

for trypsin and thrombin than for matriptase-2 inhibition. Note-

aminobutyric acid (n = 2 or 3) was found to be rather preferred

worthy, with respect to the three proteases and inhibitors 60–65,

Table 5

Synthesis and biological evaluation of benzamidines 36–59 with tert-butyl benzoate and benzoic acid substructures

1. NMM,

ClCO -Bu,

₂i

3- or 4-cyano-

benzenesulfonyl

chloride, DIPEA

amine

2. H₂, Pd/C

BuO C

NHCbz

NH₂

O₂

1. H₂NOH×HCl, DIPEA

2. Ac₂O

3. H₂, Pd/C

4. TFA (0.1 N),

TFA,

then H₂O,

NH₂

lyophilization

BuO C

HO₂C

²F₃CCO₂

36-47

Arom. subst.

48-59

Human thrombin K_i^a

Compd

CO₂t-Bu/CO₂H

Bovine trypsin K_i^a

(

lM)

(

lM)

Human matriptase-2 K_i^a

(lM)

4-CO₂t-Bu

3-CO₂t-Bu

4-CO₂t-Bu

3-CO₂t-Bu

4-CO₂t-Bu

3-CO₂t-Bu

4-CO₂t-Bu

3-CO₂t-Bu

4-CO₂t-Bu

3-CO₂t-Bu

4-CO₂t-Bu

3-CO₂t-Bu

4-CO₂H

3-CO₂H

4-CO₂H

3-CO₂H

4-CO₂H

3-CO₂H

4-CO₂H

3-CO₂H

4-CO₂H

para

meta

para

meta

10.9 0.6

26.4 0.7

7.1 0.1

174 14

100

103

115 12

13.8 0.8

7.4 0.5

n.i.

137

7.6 0.3

7.9 0.5

11.2 0.3

16.7 0.8

12.3 0.3

12.2 0.3

12.1 0.9

126

101

128 13

25.0 0.6

21.9 0.8

30.1 0.9

n.i.

139 22

207 25

n.i.

201 23

52 10

88 14

75 13

28.2 0.8

3-CO₂H

4-CO₂H

3-CO₂H

208

217 11

n.i.

For assay details, see Table 1.

6494

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

Table 6

Synthesis and biological evaluation of bisbenzamidines 60–73

1. NMM, ClCO -Bu,

₂i

2. 4-cyanobenzylamine

2. HCl

3- or 4-cyanobenzenesulfonyl

chloride, DIPEA

n-1

NH₃

n-1

NHBoc

R¹R²

C + M

1. H₂NOH×HCl, DIPEA

2. Ac₂O

O₂

n-1

3. H₂, Pd/C

NH₂

4. TFA (0.1 N), HPLC

5. lyophilization

R¹R²

H₂N

NH₂

F₃CCO₂

60-73

Compd

R¹

R²

R/S

Arom. subst.

Bovine trypsin K_i^a

(lM)

Human thrombin K_i^a

Human matriptase-2 K_i^a

(lM)

CH₂OBn

—

para

meta

para

meta

3.7 0.3

0.53 0.02

0.67 0.04

1.59 0.08

0.60 0.20

0.57 0.04

0.42 0.02

8.4 0.2

0.54 0.05

6.5 0.2

0.18 0.01

4.0 0.2

125 12

10.6 0.3

7.4 0.4

19.0 0.5

10.7 0.6

8.9 0.8

8.2 0.7

5.1 0.3

16.5 0.8

6.0 0.2

11.8 0.5

25.3 0.7

16.3 0.7

0.93 0.07

10.7 0.5

2.4 0.2

11.2 0.9

0.53 0.02

CH₂OBn

0.26 0.02

1.8 0.1

CH₂OBn

12.5 0.4

For assay details, see Table 1.

a positive impact for binding afﬁnity caused by the meta substitu-

tion was not observed.

showed similar matriptase-2 inhibition and the structure of the

hydrophobic side chain did not markedly inﬂuence the inhibitors’

potency.

Based on the structures of the glycine-connected substances 60

(para substitution) and 63 (meta substitution) we designed

branched bisbenzamidines, in which one backbone hydrogen atom

was replaced by a benzyl or benzyloxymethylene residue (n = 1;

R = Bn or CH₂OBn). The corresponding phenylalanine- and O-ben-

zylserine-connected bisbenzamidines 66–73 bear a terminal aro-

matic core to potentially increase afﬁnity to the proteases. Thus,

the active site mapping was extended by hydrophobic interactions

with putative binding areas, in addition to the polar interactions of

the two benzamidine substructures. Depending on the stereo-

chemical conﬁguration of the branched bisbenzamidines, introduc-

tion of the hydrophobic side chain either improved the activity

towards trypsin (60 and 63 vs S-conﬁgured compounds 66, 68

and 70, 72) or impaired it (60 and 63 vs R-conﬁgured compounds

67, 69 and 71, 73). In contrast, in the case of thrombin, R-conﬁg-

ured derivatives 67, 69, 71, 73 represented the eutomers and S-

conﬁgured derivatives 66, 68, 70, 72 the distomers. Each of the

meta-substituted inhibitors was slightly more potent than the cor-

responding para compound on trypsin and thrombin. Next, the

inﬂuence of the structure of the hydrophobic side chain was in-

spected. When comparing the corresponding eutomers on trypsin,

phenylalanine and O-benzylserine derivatives were equipotent

(66, 68, 70, 72). With regard to thrombin inhibition, the O-benzyl-

serine-connected eutomers 69 and 73 exhibited lower K_ivalues

3. Conclusions

An active site mapping for three related serine proteases was

performed. Throughout six series of sulfamoyl benzamidine inhib-

itors, an ascending level of complexity was achieved. We have

systematically introduced several points of diversity, including

the substitution pattern at the terminal aromatic cores, the nature

of the connecting spacer and the attachment of a branch to the

linker. A typical salt bridge between the amidino function and

the carboxylate group of Asp189 situated in the S1 cavity is as-

sumed. With a so anchored sulfamoyl benzamidine moiety at

the S1 pocket, the impact of the substituent at the second aro-

matic ring could be elucidated. This part of the molecules is ex-

pected to scan the extended S3/S4 binding site of the three

target enzymes. As an overall trend, the presence of a carboxyl

group in the second aromatic ring was clearly unfavored. Both, a

dimethoxy and a tert-butoxycarbonyl substitution led to an im-

proved afﬁnity towards trypsin, thrombin and matriptase-2. This

ﬁnding can be attributed to the known hydrophobic character of

the S3/S4 binding site of the proteases.

General observations indicate that trypsin-like enzymes favor

ligands with large hydrophobic substituents in the S3/S4 pock-

et.^35,47In trypsin, this region represents an aryl binding site. It is

formed by Leu99 and Gly174 and the ethylene side chain of

Gln175 and has Trp215 at its bottom. The N-terminal naph-

thylsulfonyl group of amidinophenylalanine amides is buried in

this pocket.^32,34In thrombin, the S3 pocket is rather ﬂat and two

aliphatic amino acids, Leu99 and Ile174, make up the S4 binding

(0.93 and 0.53

ﬂexibility. The phenylalanine-connected eutomers 67 and 71 were

less potent (6.0 and 2.4 M). Surprisingly, matriptase-2 catalysis

lM) probably owing to their larger size and higher

was hardly affected by the introduction of the third branch into

the bisbenzamidine framework (66–73). Signiﬁcant differences be-

tween the corresponding S- and R-conﬁgured enantiomers were

not detected. Moreover, para- or meta-substituted derivatives

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

6495

site which is terminated by Trp215. Thus, the cyclohexyl moiety of

melagatran orients to this region. Aromatic units also occupy the

S4 pocket of thrombin,³⁴for example the 2-pyridyl residue of

dabigatran, as well as the arylsulfonyl group of NAPAP and argatro-

ban.³²Accordingly, the structure of the complex between the irre-

in inhibitors of trypsin-like serine proteases containing an

arginine mimetic as P1 amino acid and an -amino acid at P2

position.^30–32,50A branched substructure as present in 66–73 did

not result in a beneﬁt in terms of matriptase-2 inhibition, regard-

less of the stereochemistry. Those regions of the active site of

matriptase-2 that can be reached by these hydrophobic branches

do obviously not allow for attractive interactions. Thus, polar

residues, in particular basic groups should be introduced into the

bisbenzamidine scaffold for the design of improved inhibitors.

versible inhibitor D-Phe-Pro-Arg-chloromethylketone (PPACK) and

thrombin shows the phenyl group in the S4 pocket.⁴⁸In matrip-

tase-2, the S3/S4 binding region is also hydrophobic in nature.

Trp783 (chymotrypsinogen numbering 215) is still present,

whereas the aliphatic-unpolar amino acids at positions 99 and

174 are exchanged for His665(99) and Tyr740(174). The S3/S4

pocket extents to Leu785(217) making this spot more hydrophobic

compared to trypsin or thrombin which have Ser or Glu in the cor-

responding position. The cyclohexyl ring of one of the few known

4. Experimental

4.1. General methods and materials

matriptase-2 inhibitors, that is, benzylsulfonyl-D-cyclohexylala-

Melting points were determined on a Büchi B510 oil bath appa-

ratus and are not corrected. Thin-layer chromatography was per-

formed on Merck aluminum sheets, silica gel 60 F₂₅₄. Preparative

column chromatography was performed on Acros silica gel

(0.06–0.20 mm, 60 Å). ¹H and ¹³C NMR spectra were acquired on

a Bruker Avanced DRX 500 spectrometer operating at 500 MHz

for ¹H and 125 MHz for ¹³C at 25 °C. Chemical shifts d are given

in ppm referring to the signal center using the solvent peak for ref-

erence: DMSO-d₆2.49/39.7 ppm. The NMR signals were assigned

by ¹H, ¹³C correlation spectra (HMQC, HMBC) using standard pulse

sequences. Spin multiplicities are indicated by the following sym-

bols: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), tt

(triplet of triplet), q (quartet), sept (septet), and m (multiplet).

The purity of all tested compounds was determined by HPLC–UV

obtained on an LC–MS instrument (ABSCIEX API 2000 LC–MS/MS,

nyl-proline-(4-amidinobenzyl)amide, might be positioned within

the S3/S4 pocket for favorable interactions with Trp783(215) and

Leu785(217), as has been proposed by docking studies.³⁹The

inhibitor exhibited an increased afﬁnity towards a mutated variant

of matriptase-2 in which Phe was introduced in place of

His665(99), resulting in a pronounced hydrophobic character of

the upper part of the S3/S4 pocket.⁴⁹

Besides hydrophobic and

pocket also enables polar and

–

interactions, the S3/S4

-cation contacts with correspond-

ing ligands.⁶In the complex of a bisbenzamidine inhibitor and

trypsin, the one benzamidine group resides in the S1 pocket and

the other one is anchored via a hydrogen-bonding network to the

backbone oxygens of amino acids deﬁning the S3/S4 binding site.¹¹

A similar binding mode was elucidated in a study on bisbenzami-

dines with a central isosorbide scaffold in the complexes with tryp-

sin mutants.³⁵In the case of thrombin, the S3 pocket is considered

to provide ionic contacts to ligands, whereas the S4 pocket tends to

accommodate lipophilic groups.³²The model of matriptase-2 re-

vealed a somewhat different feature in that the upper part of the

S3/S4 pocket, formed by the backbone carbonyl oxygens of

Glu662(96), Asp663(97) and Ser664(98), represents a negatively

charged environment. The guanidino group of a second matrip-

HPLC Agilent 1100). A sample of 10

into a Phenomenex Luna C18 HPLC column (50 Â 2.00 mm, particle

size 3 m) and was chromatographed at a ﬂow rate of 250 L/min

lL (0.5 mg, mL) was injected

using four different HPLC methods. Method a was as follows: The

compound was dissolved in water. Mobile phase A, water with

2 mM ammonium acetate; B, methanol; gradient, 10%

B in

20 min to 100% B, 10 min 100% B. Method b was as follows: The

compound was dissolved in methanol. Mobile phase A, water with

10 mM ammonium formiate and 0.1% formic acid; B, methanol

with 0.1% formic acid; gradient, 10% B in 20 min to 100% B,

10 min 100% B. Method c was as follows: The compound was dis-

solved in methanol. Mobile phase A, water with 10 mM ammo-

nium formiate and 0.1% formic acid; B, methanol with 0.1%

formic acid; gradient, 40% B in 20 min to 100% B, 10 min 100% B.

Method d was as follows: The compound was dissolved in water.

Mobile phase A, water with 10 mM ammonium formiate and

0.1% formic acid; B, methanol with 0.1% formic acid; gradient,

10% B in 20 min to 100% B, 10 min 100% B. UV absorption was de-

tected from 200 to 950 nm using a diode array detector. Purity of

the compounds was determined at 220–400 nm. Mass spectra

were recorded on an API 2000 mass spectrometer (electron spray

ion source, ABSCIEX, Darmstadt, Germany) coupled with an Agilent

1100 HPLC system. Elemental microanalyses were performed on a

VarioEL apparatus. Enzymatic reactions were monitored in 96 well

plates with ﬂat bottom and lid (Sarstedt, Newton, USA) on a FLUO-

star Optima ﬂuorimeter (BMG LABTECH, Offenburg, Germany). The

substrates Boc-Gln-Ala-Arg-NH-Mec (human matriptase-2, bovine

trypsin) and Cbz-Gly-Gly-Arg-NH-Mec (human thrombin) were

obtained from Bachem (Bubendorf, Switzerland). Bovine trypsin

was purchased from Sigma and human thrombin from Calbiochem

(Darmstadt, Germany). The conditioned medium of HEK-MT2 cells

was used as source for matriptase-2 as described.^38,39Commercial

available 4-cyanobenzylamine (Sigma, Steinheim, Germany), 4-

cyanobenzenesulfonyl chloride (Alfa Aesar, Karlsruhe, Germany)

and 3-cyanobenzenesulfonyl chloride (Fluorochem, Derbyshire,

Great Britain) were used without further puriﬁcation.

tase-2 inhibitor, that is, benzylsulfonyl-D-argininyl-proline-(4-

amidinobenzyl)amide, was proposed to point to this region.³⁹

The incorporation of a second benzamidine moiety was con-

ducted to study the inﬂuence of these basic substructures (60–73).

Indeed, in comparison with the acidic groups present in 48–59,

a clear overall improvement in afﬁnity was achieved. These

results reﬂect attractive interactions to the putative S3/S4 binding

region of trypsin, thrombin and matriptase-2. Following our initial

assumption on the binding mode of bisbenzamidines, the sulfa-

moyl benzamidine group is anchored in the S1 pocket and the ami-

nomethyl benzamidine group resides in the S3/S4 cavity. However,

a reversed orientation can be taken into consideration, because the

preference for a meta-substitution at the sulfamoyl benzamidine

portion is much less obvious for compounds 60–73. Such a retro-

binding mode was observed in trypsin complexes with two related

bisbenzamidine inhibitors. In one case, the sulfamoyl benzamidine

portion was inserted into the S1 pocket, whereas in the other one,

the amidinophenylalanine side chain was bound in this pocket.¹¹

Our study demonstrated unexpected opposite effects of the

stereochemistry of branched bisbenzamidines on trypsin and

thrombin inhibition. An incorporation of an S-conﬁgured, hydro-

phobic amino acid was advantageous for trypsin, whereas a con-

trary result was obtained for thrombin. The conﬁguration of

substrate-analogue inhibitors has a strong impact on their activity;

but opposite effects on trypsin and thrombin have not been

observed so far. For example, in 2-naphthylsulfonyl-(3-amidin-

ophenyl)alanine-piperazides, the

potent than the

-analogs against both, trypsin and thrombin.⁵

-conﬁgured amino acid at P3 position is generally preferred

L-enantiomers were much more

6496

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

4.2. General procedures

4.2.8. Preparation of benzamidines

A mixture of the corresponding nitrile B, E, G, I, or L, (1 mmol)

hydroxylamine hydrochloride (139 mg, 2 mmol), DIPEA (259 mg,

2 mmol) and abs ethanol (10 mL) was stirred 1 h at 87 °C. After-

wards the solvent was removed under reduced pressure, the resi-

due was taken up in acetonitrile (10 mL) and, if necessary, a

minimum amount of acetic acid was added until a nearly complete

dissolution was observed. Insoluble solids were ﬁltered off and

after addition of acetic anhydride (306 mg, 3 mmol) the solution

was stirred 1 h at room temperature. The basic reactants were sep-

arated from the desired O-acetylamidoximes by ﬂash column chro-

matography (ethyl acetate/ethanol = 10:1) and after removal of the

solvent the crude product was dissolved in acetic acid (20 mL). The

reduction was performed with 10% Pd/C under a hydrogen atmo-

sphere (3 atm) at room temperature. After ﬁltration the mixture

was evaporated under reduced pressure. The respective benzami-

dines were obtained as follows.

4.2.1. Preparation of nitriles B

A mixture of 3- or 4-cyanobenzenesulfonyl chloride (605 mg,

3 mmol), the amine (3.3 mmol) and pyridine (736 mg, 9.3 mmol)

was stirred overnight at room temperature in abs dichloromethane

(40 mL). After removal of the solvent at reduced pressure, nitriles B

were obtained by ﬂash column chromatography (dichlorometh-

ane) and recrystallisation from ethyl acetate/hexane or toluene.

4.2.2. Preparation of amines D and H

To a solution of the Boc-protected amino acid (C, 10 mmol) and

N-methylmorpholine (1.01 g, 10 mmol) in abs THF (20 mL) isobutyl

chloroformate (1.37 g, 10 mmol) was added dropwise at À30 °C.

After stirring for 15 minutes the amine (10 mmol) was added in

one portion. The mixture was stirred and allowed to reach room

temperature within 2 h. The reaction was quenched with water

(5 mL) and THF was evaporated. The residue was dissolved in ethyl

acetate (100 mL), was successively washed with KHSO₄(1 Â 25 mL),

NaHCO₃(1 Â 25 mL) and NaCl (1 Â 25 mL) and dried over Na₂SO₄.

Gaseous HCl was passed through the dried solution for 4 h and the

precipitated amine hydrochlorides D and H were ﬁltered off.

4.2.8.1. Benzamidines 1–16.

To the oily residue abs ethyl

acetate (À20 °C, 50 mL) was added upon vigorous stirring and

the precipitate, which was separated by suction ﬁltration, was dis-

solved in HCl (0.1 N, 50 mL). After ﬁltration through a syringe ﬁlter

(cellulose acetate, 0.2

after lyophilization.

lm) the benzamidines 1–16 were obtained

4.2.3. Preparation of amines N

The synthesis of amines N was performed as described for

amines D and H, but 4-cyanobenzylamine was employed as hydro-

chloride. Thus, 2 equivalents of N-methylmorpholine were used.

4.2.8.2. Benzamidines 17–28.

The oily residue was dissolved

in acetic acid (2 mL) and the solution was rapidly injected into stir-

ring ethyl acetate (À20 °C, 200 mL). Gaseous HCl was bubbled

through and the precipitate was separated by suction ﬁltration.

The solid was dissolved in HCl (0.1 N, 50 mL) and after ﬁltration

through a syringe ﬁlter (cellulose acetate, 0.2

dines 17–28 were obtained after lyophilization.

4.2.4. Preparation of amines F and K

Amines F and K were synthesized according to the preparation

of amines D and H using Cbz-protected amino acids. After extrac-

tion the crude product was puriﬁed by column chromatography

(petroleum ether/ethyl acetate = 6:1) to separate the isobutyl car-

bamate by-product. The desired product was then eluated from

the column using ethyl acetate as the solvent. After reducing the

solvent to a volume of 300 mL, the Cbz-protected amine was re-

duced with 10% Pd/C under a hydrogen atmosphere (3 atm) to af-

ford the amines F and K after ﬁltration and evaporation of the

solvent.

m) the benzami-

4.2.8.3. Benzamidines 29–32, 33–35 and 36–47.

To the oily

residue abs ethyl acetate (À20 °C, 50 mL) was added and after stir-

ring for 1 h the precipitate, which was separated by suction ﬁltra-

tion, was dissolved in triﬂuoroacetic acid (0.1 N, 50 mL). After

ﬁltration through a syringe ﬁlter (cellulose acetate, 0.2 lm) the

benzamidines 29–32, 33–35 and 36–47 were obtained after

lyophilization.

4.2.5. Preparation of nitriles E and I

A solution of 3- or 4-cyanobenzenesulfonyl chloride (605 mg,

3 mmol), the amine hydrochlorides D and H (3 mmol) and DIPEA

(776 mg, 6 mmol) in THF (30 mL) was stirred overnight at room

temperature. After removing of the solvent, the crude product

was dissolved in ethyl acetate (100 mL) and the mixture was

washed with KHSO₄(1 Â 50 mL), NaCl (1 Â 50 mL) and was dried

over Na₂SO₄. After evaporation of the solvent, nitriles E and I were

obtained by recrystallisation from ethyl acetate/hexane or ethyl

acetate/toluene.

4.2.8.4. Benzamidines 48–59.

The corresponding benzami-

dines 36–47 (100 mg) were stirred in triﬂuoroacetic acid (6 mL)

for 6 h at room temperature. Afterwards, either 50 ml of water

were added or the addition of water was stopped when a slight

precipitation appeared. Benzamidines 48–59 were obtained after

ﬁltration through a syringe ﬁlter (cellulose acetate, 0.2

lowed by lyophilization.

lm) fol-

4.2.9. Preparation of bisbenzamidines

A mixture of the corresponding dintrile O (1 mmol), hydroxyl-

amine hydrochloride (556 mg, 8 mmol), DIPEA (1.03 g, 8 mmol)

and abs ethanol (30 mL) was stirred at 87 °C overnight. Afterwards

the solvent was removed in vacuo and the crude product was puri-

ﬁed by ﬂash column chromatography (ethyl acetate) after dissolu-

tion in acetic acid (3 mL). The residue was taken up in acetonitrile

(20 mL) and an appropriate amount of acetic acid was added until a

nearly complete solution was observed. Insoluble solids were re-

moved by ﬁltration. Acetic anhydride (1.23 g, 12 mmol) was added

and the mixture was stirred at room temperature overnight. After

reducing the volume to 20 mL, 10% Pd/C was added and the reduc-

tion was carried out under a hydrogen atmosphere (3 atm) at room

temperature overnight. After ﬁltration and evaporation of the sol-

vent to a volume of 4 mL, the bisbenzamidinium diacetate was pre-

cipitated by dropwise injection of the solution into ethyl acetate

(À20 °C, 100 mL). The solid was isolated by suction ﬁltration and

4.2.6. Preparation of nitriles G and L

A solution of 3- or 4-cyanobenzenesulfonyl chloride (605 mg,

3 mmol), the amines F or K (3 mmol) and DIPEA (388 mg, 3 mmol)

in THF (30 mL) was stirred 1 h at room temperature. After evapo-

ration of the solvent and addition of acetic acid (2 mL) the nitriles

G and L were obtained by ﬂash column chromatography (dichloro-

methane/ethyl acetate = 10:1).

4.2.7. Preparation of nitriles O

A solution of 3- or 4-cyanobenzenesulfonyl chloride (605 mg,

3 mmol), the amine hydrochlorides

(776 mg, 6 mmol) in THF (30 mL) was stirred 1 h at room temper-

ature. After evaporation of the solvent, acetic acid was added

(2 mL) and the nitriles O were obtained by ﬂash column chroma-

tography (ethyl acetate).

N (3 mmol) and DIPEA

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

6497

was dissolved in triﬂuoroacetic acid (0.1 N, 50 mL). The respective

bisbenzamidines were obtained as follows.

131.6, 145.4, 158.7, 165.0. LC–MS (ESI) purity 97%, m/z 320.1

[M+H]⁺, (method a). Anal (C₁₅H₁₈ClN₃O₃S Â 0.5 H₂O) C, H, N.

4.2.9.1. Bisbenzamidines 60–65.

65 were puriﬁed by preparative reversed-phase HPLC (10%

The bisbenzamidines 60–

4.3.7. 4-(N-(3-Methoxybenzyl)sulfamoyl)benzamidinium

chloride (7)

MeOH/90% H₂O) and were obtained after lyophilization.

Yield 32%, mp 186–188 °C. ¹H NMR (DMSO-d₆): d 3.69 (s, 3H),

4.01 (d, J = 6.3 Hz, 2H), 6.77–6.82 (m, 3H), 7.17–7.21 (m, 1H),

7.98, 8.01 (each d, J = 8.2 Hz, 4H), 8.50 (t, J = 6.3 Hz, 1H), 9.43,

9.59 (each s, 4H). APT ¹³C NMR (DMSO-d₆): d 46.2, 55.2, 112.9,

113.3, 119.8, 126.9, 129.3, 129.5, 139.1, 145.4, 159.4, 165.0. LC–

MS (ESI) purity 97%, m/z 320.1 [M+H]⁺, (method a). Anal

(C₁₅H₁₈ClN₃O₃S Â 0.3 H₂O) C, H, N.

4.2.9.2. Bisbenzamidines 66–73.

The bisbenzamidines 66–

73 were puriﬁed by preparative reversed-phase HPLC (20%

MeOH/80% H₂O) and were obtained after lyophilization.

4.3. Compounds 1–73 prepared

4.3.1. 4-(N-Phenylsulfamoyl)benzamidinium chloride (1)

Yield 48%, mp 126–130 °C. ¹H NMR (DMSO-d₆): d 7.04 (tt,

J = 7.3 Hz, J = 1.3 Hz, 1H), 7.14 (dd, J = 8.7 Hz, J = 1.3 Hz, 2H), 7.23

(dd, J = 7.3 Hz, J = 8.5 Hz, 2H), 7.94, 7.97 (each d, J = 9.0 Hz, 4H),

9.41, 9.55 (each s, 4H), 10.66 (s, 1H). APT ¹³C NMR (DMSO-d₆): d

120.3, 124.5, 127.1, 129.4, 129.5, 132.3, 137.4, 144.0, 165.1. LC–

MS (ESI) purity 96%, m/z 276.4 [M+H]⁺, (method a). Anal

(C₁₃H₁₄ClN₃O₂S Â 0.1 H₂O) C, H, N.

4.3.8. 4-(N-(3,4-Dimethoxybenzyl)sulfamoyl)benzamidinium

chloride (8)

Yield 54%, mp 124–127 °C. ¹H NMR (DMSO-d₆): d 3.67, 3.69

(each s, 6H), 3.96 (d, J = 6.3 Hz, 2H), 6.72 (dd, J = 8.2 Hz, J = 1.9 Hz,

1H), 6.80 (d, J = 1.9 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 7.97, 7.99 (each

d, J = 8.8 Hz, 4H), 8.41 (t, J = 6.2 Hz, 1H), 9.41, 9.57 (each s, 4H). APT

¹³C NMR (DMSO-d₆): d 46.2, 55.6, 55.7, 111.7, 111.8, 120.0, 126.9,

129.3, 129.7, 131.6, 145.5, 148.2, 148.7, 164.9. LC–MS (ESI) purity

95%, m/z 350.3 [M+H]⁺, (method a). Anal (C₁₆H₂₀ClN₃O₄S Â 0.7

H₂O) C, H, N.

4.3.2. 4-(N-(4-Methoxyphenyl)sulfamoyl)benzamidinium

chloride (2)

Yield 53%, mp 238–239 °C. ¹H NMR (DMSO-d₆): d 3.66 (s, 3H),

6.80 (d, J = 9.2 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 7.88, 7.94 (each d,

J = 8.5 Hz, 4H), 9.39, 9.54 (each s, 4H), 10.29 (s, 1H). APT ¹³C NMR

(DMSO-d₆): d 55.3, 114.6, 123.6, 127.2, 129.4, 129.8, 132.1, 144.1,

156.8, 165.1. LC–MS (ESI) purity 97%, m/z 306.5 [M+H]⁺, (method

a). Anal (C₁₄H₁₆ClN₃O₃S Â 0.1 H₂O) C, H, N.

4.3.9. 3-(N-Phenylsulfamoyl)benzamidinium chloride (9)

Yield 75%, mp 262–263 °C. ¹H NMR (DMSO-d₆): d 7.02–7.05 (m,

1H), 7.11–7.13 (m, 2H), 7.22 (dd, J = 7.5 Hz, J = 7.3 Hz, 2H), 7.77 (dd,

J = 7.9 Hz, J = 7.9 Hz, 1H), 8.00–8.06 (m, 2H), 8.26 (dd, J = 1.7 Hz,

J = 1.6 Hz, 1H), 9.40, 9.61 (each s, 4H), 10.55 (s, 1H). APT ¹³C NMR

(DMSO-d₆): d 120.6, 124.6, 126.7, 129.3, 129.3, 130.2, 131.3,

132.7, 137.3, 164.9. LC–MS (ESI) purity 95%, m/z 276.3 [M+H]⁺,

(method a). Anal (C₁₃H₁₄ClN₃O₂S) C, H, N.

4.3.3. 4-(N-(3-Methoxyphenyl)sulfamoyl)benzamidinium

chloride (3)

Yield 48%, mp 204–205 °C. ¹H NMR (DMSO-d₆): d 3.66 (s, 3H),

6.61 (ddd, J = 8.1 Hz, J = 2.2 Hz, J = 1.0 Hz, 1H), 6.71–6.75 (m, 2H),

7.13 (dd, J = 8.2 Hz, J = 8.4 Hz, 1H), 7.95, 7.98 (each d, J = 8.8 Hz,

4H), 9.42, 9.55 (each s, 4H), 10.68 (s, 1H). APT ¹³C NMR (DMSO-

d₆): d 55.2, 106.0, 109.5, 112.1, 127.1, 129.5, 130.3, 132.5, 138.6,

144.0, 159.8, 165.1. LC–MS (ESI) purity 94%, m/z 306.4 [M+H]⁺,

(method a). Anal (C₁₄H₁₆ClN₃O₃S Â 0.1 H₂O) C, H, N.

4.3.10. 3-(N-(4-Methoxyphenyl)sulfamoyl)benzamidinium

chloride (10)

Yield 58%, mp 230–232 °C. ¹H NMR (DMSO-d₆): d 3.65 (s, 3H),

6.79 (d, J = 9.1 Hz, 2H), 7.01 (d, J = 9.2 Hz, 2H), 7.76 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 8.02, 7.96 (d, J = 7.9 Hz, 2H), 8.22 (dd, 1H,

J = 1.6 Hz, J = 1.6 Hz), 9.39, 9.60 (each s, 4H), 8.33 (s, 1H). APT ¹³C

NMR (DMSO-d₆): d 55.3, 114.5, 124.0, 126.7, 129.3, 129.7, 130.1,

131.4, 132.5, 140.5, 156.9, 165.0. LC–MS (ESI) purity 95%, m/z

306.5 [M+H]⁺, (method a). Anal (C₁₄H₁₆ClN₃O₃S) C, H, N.

4.3.4. 4-(N-(3,4-Dimethoxyphenyl)sulfamoyl)benzamidinium

chloride (4)

Yield 64%, mp 137–141 °C. ¹H NMR (DMSO-d₆): d 3.63, 3.65

(each s, 6H), 6.60 (dd, J = 8.5 Hz, J = 2.6 Hz, 1H), 6.73 (d, J = 2.5 Hz,

1H), 6.79 (d, J = 8.8 Hz, 1H), 7.91, 7.94 (each d, J = 8.8 Hz, 4H),

9.38, 9.54 (each s, 4H), 10.29 (s, 1H). APT ¹³C NMR (DMSO-d₆): d

55.6, 55.8, 106.6, 112.3, 113.7, 127.2, 129.4, 130.2, 132.1, 144.0,

146.4, 149.0, 165.1. LC–MS (ESI) purity 99%, m/z 336.0 [M+H]⁺,

(method a). Anal (C₁₅H₁₈ClN₃O₄S Â 0.4 H₂O) C, H, N.

4.3.11. 3-(N-(3-Methoxyphenyl)sulfamoyl)benzamidinium

chloride (11)

Yield 66%, mp 184–186 °C. ¹H NMR (DMSO-d₆): d 3.65 (s, 3H),

6.61 (ddd, J = 8.4 Hz, J = 2.5 Hz, J = 1.0 Hz, 1H), 6.70–6.74 (m, 2H),

7.12 (dd, J = 8.2 Hz, J = 8.2 Hz, 1H), 7.78 (dd, J = 8.2 Hz, J = 7.9 Hz,

1H), 8.01–8.07 (m, 2H), 8.27 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 9.40,

9.61 (each s, 4H), 10.54 (s, 1H). APT ¹³C NMR (DMSO-d₆): d 55.2,

106.5, 109.7, 112.6, 126.7, 129.4, 130.2, 130.2, 131.3, 132.7,

138.6, 140.5, 159.8, 165.0. LC–MS (ESI) purity 94%, m/z 306.5

[M+H]⁺, (method a). Anal (C₁₄H₁₆ClN₃O₃S Â 0.1 H₂O) C, H, N.

4.3.5. 4-(N-Benzylsulfamoyl)benzamidinium chloride (5)

Yield 13%, mp 238–239 °C. ¹H NMR (DMSO-d₆): d 4.03 (d,

J = 6.3 Hz, 2H), 7.20–7.31 (m, 5H), 7.97–8.02 (m, 4H), 8.51 (t,

J = 6.3 Hz, 1H), 9.44, 9.60 (each s, 4H). APT ¹³C NMR (DMSO-d₆): d

46.3, 127.4, 126.9, 127.7, 128.4, 129.4, 131.7, 137.5, 145.4, 165.0.

LC–MS (ESI) purity 99%, m/z 290.4 [M+H]⁺, (method a). Anal

(C₁₄H₁₆ClN₃O₂S) C, H, N.

4.3.12. 3-(N-(3,4-Dimethoxyphenyl)sulfamoyl)benzamidinium

chloride (12)

Yield 51%, mp 205–206 °C. ¹H NMR (DMSO-d₆): d 3.61, 3.65

(each s, 6H), 6.59 (dd, J = 8.8, Hz, J = 2.6 Hz, 1H), 6.71 (d,

J = 2.6 Hz, 1H), 6.78 (d, J = 8.8 Hz, 1H), 7.77 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 7.97–8.03 (m, 2H, 4-H), 8.22 (dd, J = 1.6 Hz,

J = 1.6 Hz, 1H), 9.35, 9.59 (each s, 4H), 10.15 (s, 1H). APT ¹³C NMR

(DMSO-d₆): d 55.6, 55.8, 107.3, 112.2, 114.3, 126.7, 129.3, 130.1,

130.1, 131.5, 132.5, 140.4, 146.6, 148.9, 164.9. LC–MS (ESI) purity

95%, m/z 336.0 [M+H]⁺, (method a). Anal (C₁₅H₁₈ClN₃O₄S) C, H, N.

4.3.6. 4-(N-(4-Methoxybenzyl)sulfamoyl)benzamidinium

chloride (6)

Yield 47%, mp 107–110 °C. ¹H NMR (DMSO-d₆): d 3.70 (s, 3H),

3.95 (s, 2H), 6.83 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 7.96,

7.99 (each d, J = 9.0 Hz, 4H), 8.41 (s, 1H), 9.50 (s, 4H). APT ¹³C

NMR (DMSO-d₆): d 45.8, 55.2, 113.8, 126.9, 129.1, 129.3, 129.4,

6498

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

4.3.13. 3-(N-Benzylsulfamoyl)benzamidinium chloride (13)

Yield 41%, mp 207–209 °C. ¹H NMR (DMSO-d₆): d 4.03 (d, 2H,

J = 6.3 Hz), 7.17–7.30 (m, 5H), 7.79 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H),

8.03–8.10 (m, 2H), 8.23 (dd, J = 1.7 Hz, J = 1.6 Hz, 1H), 8.43 (t,

J = 6.3 Hz, 1H), 9.42, 9.63 (each s, 4H). APT ¹³C NMR (DMSO-d₆): d

126.5, 127.3, 127.8, 128.3, 129.1, 130.2, 131.3, 132.0, 137.6,

141.7, 165.0. LC–MS (ESI) purity 97%, m/z 290.3 [M+H]⁺, (method

a). Anal (C₁₄H₁₆ClN₃O₂S) C, H, N.

6.72 (dd, J = 8.2 Hz, J = 1.9 Hz, 1H), 6.84–6.86 (m, 2H), 7.96, 8.00

(each d, J = 8.7 Hz, 4H), 8.31–8.35 (m, 2H), 9.42, 9.46 (each s, 4H).

APT ¹³C NMR (DMSO-d₆): d 42.0, 45.3, 55.6, 55.8, 111.6, 111.9,

119.6, 127.1, 129.3, 131.6, 132.0, 145.1, 148.0, 148.8, 165.1,

167.4. LC–MS (ESI) purity 95%, m/z 407.1 [M+H]⁺, (method a). Anal

(C₁₈H₂₃ClN₄O₅S Â 1.3 H₂O) C, H, N.

4.3.20. 4-(N-(4-(3,4-Dimethoxyphenylamino)-4-oxobutyl)-

sulfamoyl)benzamidinium chloride (20)

Yield 65%, mp 265 °C (Zers.). ¹H NMR (DMSO-d₆): d 1.70 (tt,

J = 7.3 Hz, J = 7.3 Hz, 2H), 2.29 (t, J = 7.3 Hz, 2H), 2.80–2.84 (m,

2H), 3.69, 3.69 (each s, 6H), 6.84 (d, J = 8.5 Hz, 1H), 7.07 (dd,

J = 8.7 Hz, J = 2.5 Hz, 1H), 7.29 (d, J = 2.6 Hz, 1H), 7.99 (s, 4H), 8.02

(t, J = 5.7 Hz, 1H), 9.37, 9.56 (each s, 4H), 9.85 (s, 1H). APT ¹³C

NMR (DMSO-d₆): d 25.3, 33.3, 42.4, 55.5, 55.9, 104.6, 111.1,

112.3, 126.9, 129.5, 131.8, 133.1, 144.8, 145.1, 148.6, 165.1,

170.2. LC–MS (ESI) purity 98%, m/z 421.4 [M+H]⁺, (method a). Anal

(C₁₉H₂₅ClN₄O₅S Â 0.4 H₂O) C, H, N.

4.3.14. 3-(N-(4-Methoxybenzyl)sulfamoyl)benzamidinium

chloride (14)

Yield 63%, mp 186–187 °C. ¹H NMR (DMSO-d₆): d 3.70 (s, 3H),

3.95 (d, J = 6.0 Hz, 2H), 6.80 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.8 Hz,

2H), 7.79 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 8.03–8.09 (m, 2H), 8.22

(dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 8.33 (t, J = 6.3 Hz, 1H), 9.43, 9.63

(each s, 4H). APT ¹³C NMR (DMSO-d₆): d 45.8, 55.2, 113.8, 126.5,

129.0, 129.4, 129.2, 130.2, 131.3, 132.0, 141.8, 158.6, 164.9. LC–

MS (ESI) purity 96%, m/z 320.3 [M+H]⁺, (method a). Anal

(C₁₅H₁₈ClN₃O₂S) C, H, N.

4.3.21. 4-(N-(3-(3,4-Dimethoxybenzylamino)-3-oxopropyl)

sulfamoyl)benzamidinium chloride (21)

4.3.15. 3-(N-(3-Methoxybenzyl)sulfamoyl)benzamidinium

chloride (15)

Yield 45%, mp 164–166 °C. ¹H NMR (DMSO-d₆): d 2.34 (t,

J = 7.3 Hz, 2H), 2.96–3.00 (m, 2H), 3.70, 3.71 (each s, 6H), 4.14 (d,

J = 5.7 Hz, 2H), 6.74 (dd, J = 8.2 Hz, J = 2.2 Hz, 1H), 6.84 (d,

J = 1.9 Hz, 1H), 6.85 (d, J = 8.5 Hz), 8.00, 8.00 (each s, 4H), 8.04 (t,

J = 6.0 Hz, 1H), 8.38 (t, J = 5.7 Hz, 1H), 9.44, 9.61 (each s, 4H). APT

¹³C NMR (DMSO-d₆): d 35.7, 39.4, 42.0, 55.6, 55.8, 111.6, 111.9,

119.6, 127.0, 129.5, 131.9, 132.0, 144.8, 147.9, 148.8, 165.2,

169.6. LC–MS (ESI) purity 97%, m/z 421.5 [M+H]⁺, (method a). Anal

(C₁₉H₂₅ClN₄O₅S Â 1.9 H₂O) C, H, N.

Yield 80%, mp 104–109 °C. ¹H NMR (DMSO-d₆): d 3.68 (s, 3H),

4.01 (d, J = 6.3 Hz, 2H), 6.74–6.82 (m, 3H), 7.79 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 8.01–8.09 (m, 2H), 8.20–8.24 (m, 1H), 8.41 (t,

J = 6.3 Hz, 1H), 9.39, 9.60 (each s, 4H). APT ¹³C NMR (DMSO-d₆): d

46.2, 55.1, 112.8, 113.3, 119.9, 126.5, 129.0, 129.4, 130.2, 131.3,

131.9, 139.1, 141.8, 159.3, 164.9. LC–MS (ESI) purity 98%, m/z

320.0 [M+H]⁺, (method a). Anal (C₁₅H₁₈ClN₃O₃S Â 0.9 H₂O) C, H, N.

4.3.16. 3-(N-(3,4-Dimethoxybenzyl)sulfamoyl)benzamidinium

chloride (16)

Yield 58%, mp 122–131 °C. ¹H NMR (DMSO-d₆): d 3.64, 3.69

(each s, 6H), 3.96 (s, 2H), 6.73 (dd, J = 8.2 Hz, J = 1.9 Hz, 1H), 6.80

(d, J = 1.9 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 7.77 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 8.02–8.07 (m, 2H), 8.23 (dd, J = 1.6 Hz, J = 1.7 Hz,

1H), 8.33 (s, 1H), 9.45, 9.57 (each s, 4H). APT ¹³C NMR (DMSO-

d₆): d 46.2, 55.5, 55.7, 111.6, 111.8, 120.1, 126.5, 128.9, 129.7,

130.1, 131.3, 131.9, 141.9, 148.2, 148.7, 164.8. LC–MS (ESI) purity

96%, m/z 350.1 [M+H]⁺. Anal (C₁₆H₂₀ClN₃O₄S Â 0.5 H₂O) C, H, N.

4.3.22. 4-(N-(4-(3,4-Dimethoxybenzylamino)-4-oxobutyl)-

sulfamoyl)benzamidinium chloride (22)

Yield 48%, mp 134–140 °C. ¹H NMR (DMSO-d₆): d 1.64 (tt,

J = 7.3 Hz, J = 7.3 Hz, 2H), 2.12 (t, J = 7.3 Hz, 2H), 2.76–2.80 (m,

2H), 3.70, 3.70 (each s, 6H), 4.14 (d, J = 5.7 Hz, 2H), 6.72 (dd,

J = 8.2 Hz, J = 1.9 Hz, 1H), 6.83 (d, J = 2.2 Hz, 1H), 6.85 (d,

J = 8.2 Hz), 7.98, 8.01 (each d, J = 8.7 Hz, 4H), 7.97–8.02 (m, 1H),

8.29 (t, J = 5.7 Hz, 1H), 9.45, 9.61 (each s, 4H). APT ¹³C NMR

(DMSO-d₆): d 25.6, 32.5, 41.9, 42.4, 55.6, 55.8, 111.6, 111.9,

119.5, 126.9, 129.5, 131.8, 132.2, 145.2, 147.9, 148.8, 165.2,

171.5. LC–MS (ESI) purity 99%, m/z 435.6 [M+H]⁺, (method a). Anal

(C₂₀H₂₇ClN₄O₅S Â 1.5 H₂O) C, H, N.

4.3.17. 4-(N-(2-(3,4-Dimethoxyphenylamino)-2-oxoethyl)-

sulfamoyl)benzamidinium chloride (17)

Yield 47%, mp 140–150 °C. ¹H NMR (DMSO-d₆): d 3.68, 3.69

(each s, 6H), 3.71 (d, J = 6.0 Hz, 2H), 6.84 (d, J = 8.8 Hz, 1H), 7.02

(dd, J = 8.7 Hz, J = 2.5 Hz, 1H), 7.18 (d, J = 2.5 Hz, 1H), 7.99, 8.02

(each d, J = 8.8 Hz, 4H), 8.42 (t, J = 6.0 Hz, 1H), 9.37, 9.56 (each s,

4H), 10.00 (s, 1H). APT ¹³C NMR (DMSO-d₆): d 45.9, 55.6, 55.9,

104.8, 111.5, 112.3, 127.1, 129.3, 131.7, 132.3, 145.2, 145.3,

148.7, 165.0, 165.8. LC–MS (ESI) purity 96%, m/z 393.3 [M+H]⁺,

(method a). Anal (C₁₇H₂₁ClN₄O₅S Â 1.0 H₂O) C, H, N.

4.3.23. 3-(N-(2-(3,4-Dimethoxyphenylamino)-2-oxoethyl)-

sulfamoyl)benzamidinium chloride (23)

Yield 30%, mp 98–104 °C. ¹H NMR (DMSO-d₆): d 3.68, 3.69 (each

s, 6H), 3.71 (d, J = 5.7 Hz, 2H), 6.84 (d, J = 8.8 Hz, 1H), 7.03 (dd,

J = 8.7 Hz, J = 2.5 Hz, 1H), 7.20 (d, J = 2.6 Hz, 1H), 7.81 (dd,

J = 7.9 Hz, J = 7.9 Hz, 1H), 8.01–8.03 (m, 1H), 8.13–8.15 (m, 1H),

8.26–8.31 (m, 2H, 2-H), 9.35, 9.62 (each s, 4H), 10.04 (s, 1H). APT

¹³C NMR (DMSO-d₆): d 45.9, 55.5, 55.9, 104.7, 111.4, 112.2, 126.6,

129.0, 130.2, 131.5, 132.2, 132.3, 141.4, 145.1, 148.7, 164.9,

165.9. LC–MS (ESI) purity 96%, m/z 393.3 [M+H]⁺, (method a). Anal

(C₁₇H₂₁ClN₄O₅S Â 2.0 H₂O) C, H, N.

4.3.18. 4-(N-(3-(3,4-Dimethoxyphenylamino)-3-oxopropyl)-

sulfamoyl)benzamidinium chloride (18)

Yield 51%, mp 150–170 °C. ¹H NMR (DMSO-d₆): d 2.45–2.50 (m,

2H), 3.03–3.07 (m, 2H), 3.69, 3.69 (each s, 6H), 6.84 (d, J = 8.9 Hz,

1H), 7.07 (dd, J = 8.7 Hz, J = 2.5 Hz, 1H), 7.31 (d, J = 2.3 Hz, 1H),

8.00 (s, 4H), 8.10 (t, J = 5.7 Hz, 1H), 9.43, 9.60 (each s, 4H), 9.96

(s, 1H). APT ¹³C NMR (DMSO-d₆): d 36.5, 39.1, 55.5, 55.9, 104.6,

111.2, 112.2, 127.0, 129.4, 131.9, 132.9, 144.8, 144.9, 148.6,

165.2, 168.3. LC–MS (ESI) purity 96%, m/z 407.3 [M+H]⁺, (method

a). Anal (C₁₈H₂₃ClN₄O₅S Â 1.5 H₂O) C, H, N.

4.3.24. 3-(N-(3-(3,4-Dimethoxyphenylamino)-3-oxopropyl)-

sulfamoyl)benzamidinium chloride (24)

Yield 63%, mp 92–100 °C. ¹H NMR (DMSO-d₆): d 2.45–2.50 (m,

2H), 3.04–3.08 (m, 2H), 3.69, 3.69 (each s, 6H), 6.84 (d, J = 8.9 Hz,

1H), 7.06 (dd, J = 8.7 Hz, J = 2.6 Hz, 1H), 7.30 (d, J = 2.5 Hz, 1H),

7.83 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 8.00 (t, J = 5.7 Hz, 1H), 8.04–

8.06 (m, 1H), 8.11–8.13 (m, 1H), 8.25 (dd, J = 1.9 Hz, J = 1.9 Hz,

1H), 9.38, 9.62 (each s, 4H), 9.93 (s, 1H). APT ¹³C NMR (DMSO-

d₆): d 36.6, 39.1, 55.5, 55.9, 104.6, 111.2, 112.2, 126.5, 129.3,

130.2, 131.3, 132.1, 132.9, 141.3, 144.9, 148.6, 165.1, 168.3. LC–

4.3.19. 4-(N-(2-(3,4-Dimethoxybenzylamino)-2-oxoethyl)-

sulfamoyl)benzamidinium chloride (19)

Yield 8%, mp 164–166 °C. ¹H NMR (DMSO-d₆): d 3.53 (d,

J = 6.0 Hz, 2H), 3.71, 3.72 (each s, 6H), 4.15 (d, J = 6.0 Hz, 2H),

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

6499

MS (ESI) purity 95%, m/z 407.3 [M+H]⁺, (method a). Anal

(C₁₈H₂₃ClN₄O₅S Â 2.2 H₂O) C, H, N.

J = 31.9 Hz), 165.0, 166.3. LC–MS (ESI) purity 98%, m/z 333.1

[M+H]⁺, (method b). Anal (C₁₇H₁₇F₃N₄O₅S Â 1.0 H₂O) C, H, N.

4.3.25. 3-(N-(2-(3,4-Dimethoxybenzylamino)-2-oxoethyl)-

sulfamoyl)benzamidinium chloride (25)

4.3.30. 3-(N-(2-(Naphthalen-2-ylamino)-2-oxoethyl)sulfamoyl)

benzamidinium triﬂuoroacetate (30)

Yield 29%, mp 120–124 °C. ¹H NMR (DMSO-d₆): d 3.53 (d,

J = 5.7 Hz, 2H), 3.71, 3.71 (each s, 6H), 4.11 (d, J = 6.0 Hz, 2H),

6.71 (dd, J = 8.2 Hz, J = 1.9 Hz, 1H), 6.84 (d, J = 1.9 Hz, 1H), 6.84 (d,

J = 8.2 Hz, 1H), 7.80 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 8.03–8.05 (m,

1H), 8.11–8.13 (m, 1H), 8.25 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 8.29

(t, J = 6.3 Hz, 1H), 8.41 (t, J = 6.0 Hz), 9.41, 9.63 (each s, 4H). APT

¹³C NMR (DMSO-d₆): d 42.0, 45.4, 55.6, 55.8, 111.6, 111.9, 119.4,

126.6, 129.1, 130.2, 131.5, 132.2, 131.6, 141.2, 147.9, 148.8,

165.0, 167.6. LC–MS (ESI) purity 95%, m/z 407.1 [M+H]⁺, (method

a). Anal (C₁₇H₂₁ClN₄O₅S Â 1.6 H₂O) C, H, N.

Yield 64%, mp 216–220 °C. ¹H NMR (DMSO-d₆): d 3.83 (s, 2H),

7.40 (ddd, J = 7.4 Hz, J = 7.4 Hz, J = 1.0 Hz, 1H), 7.46 (ddd,

J = 7.4 Hz, J = 7.4 Hz, J = 1.3 Hz, 1H), 7.52 (dd, J = 8.8 Hz, J = 2.2 Hz,

1H), 7.78–7.86 (m, 4H), 8.01–8.03 (m, 1H), 8.14 (d, J = 1.6 Hz),

8.17–8.19 (m, 1H), 8.24 (dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 9.24 (s,

4H). APT ¹³C NMR (DMSO-d₆): d 46.0, 115.6, 120.0, 126.4, 124.9,

126.6, 127.4, 127.6, 128.6, 130.0, 130.2, 131.5, 132.1, 133.4,

136.2, 141.6, 158.7 (q, J = 31.9 Hz), 165.0, 166.7. LC–MS (ESI) purity

95%, m/z 383.3 [M+H]⁺, (method b). Anal (C₂₁H₁₉F₃N₄O₅S) C, H, N.

4.3.31. 3-(N-(2-Cyclohexylamino)-2-oxoethyl)-N-sulfamoyl)-

benzamidinium triﬂuoroacetate (31)

4.3.26. 3-(N-(4-(3,4-Dimethoxyphenylamino)-4-oxobutyl)-

sulfamoyl)benzamidinium chloride (26)

Yield 73%, mp 194–199 °C. ¹H NMR (DMSO-d₆): d 1.04–1.25 (m,

5H), 1.49–1.63 (m, 5H), 3.36–3.43 (m, 1H), 3.49 (d, J = 6.3 Hz, 2H),

7.68 (d, J = 7.9 Hz, 1H), 7.82 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 8.01–

8.03 (m, 1H), 8.09–8.12 (m, 2H, 4-H), 8.16 (dd, J = 1.9 Hz,

J = 1.9 Hz, 1H), 9.51 (s, 4H). APT ¹³C NMR (DMSO-d₆): d 24.5,

25.3, 32.3, 45.3, 47.7, 126.4, 129.3, 130.2, 131.5, 132.1, 141.5,

158.9 (q, J = 31.9 Hz), 165.1, 166.3. LC–MS (ESI) purity 99%, m/z

339.4 [M+H]⁺. Anal (C₁₇H₂₃F₃N₄O₅S Â 0.7 H₂O) C, H, N.

Yield 65%, mp 123–125 °C. ¹H NMR (DMSO-d₆): d 1.69 (tt,

J = 7.3 Hz, J = 7.3 Hz, 2H), 2.29 (t, J = 7.3 Hz, 2H), 2.79–2.82 (m,

2H), 3.69, 3.69 (each s, 6H), 6.84 (d, J = 8.9 Hz, 1H), 7.07 (dd,

J = 8.7 Hz, J = 2.3 Hz, 1H), 7.30 (d, J = 2.3 Hz, 1H), 7.82 (dd,

J = 7.9 Hz, J = 7.9 Hz, 1H), 7.94 (t, J = 5.7 Hz, 1H), 8.04–8.06 (m,

1H), 8.10–8.12 (m, 1H), 8.25 (s, 1H), 9.41, 9.64 (each s, 4H), 9.88

(s, 1H). APT ¹³C NMR (DMSO-d₆): d 25.3, 33.3, 42.3, 55.5, 55.9,

104.6, 111.1, 112.3, 126.5, 129.3, 130.2, 131.3, 132.1, 133.1,

141.4, 144.8, 148.6, 165.1, 170.3. LC–MS (ESI) purity 99%, m/z

421.3 [M+H]⁺, (method a). Anal (C₁₉H₂₅ClN₄O₅S Â 1.1 H₂O) C, H, N.

4.3.32. 3-(N-(2-Isopropylamino)-2-oxoethyl)-N-

sulfamoyl)benzamidinium triﬂuoroacetate (32)

Yield 64%, mp 80–90 °C. ¹H NMR (DMSO-d₆):

d 0.96 (d,

4.3.27. 3-(N-(3-(3,4-Dimethoxybenzylamino)-3-oxopropyl)-

sulfamoyl)benzamidinium chloride (27)

J = 6.7 Hz, 6H), 3.47 (d, J = 6.3 Hz, 2H), 3–66–3.73 (m, 1H), 7.67

(d, J = 7.9 Hz, 1H), 7.82 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 8.01–8.03

(m, 1H), 8.09–8.12 (m, 2H), 8.17 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H),

9.42, 9.49 (each s, 4H). APT ¹³C NMR (DMSO-d₆): d 22.3, 40.6,

45.3, 126.4, 129.3, 130.2, 131.5, 132.1, 141.5, 158.7 (q,

J = 31.9 Hz), 165.0, 166.3. LC–MS (ESI) purity 97%, m/z 299.4

[M+H]⁺, (method b). Anal (C₁₄H₁₉F₃N₄O₅S Â 0.9 H₂O) C, H, N.

Yield 53%, mp 132–138 °C. ¹H NMR (DMSO-d₆): d 2.33 (t,

J = 7.3 Hz, 2H), 2.97–3.01 (m, 2H), 3.70, 3.71 (each s, 6H), 4.14 (d,

J = 5.7 Hz, 2H), 6.73 (dd, J = 8.1 Hz, J = 1.9 Hz, 1H), 6.83 (d,

J = 1.9 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 7.82 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 7.95 (t, J = 5.7 Hz, 1H), 8.05–8.07 (m, 1H), 8.10–

8.12 (m, 1H), 8.25 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 8.38 (t,

J = 5.7 Hz), 9.44, 9.65 (each s, 4H). APT ¹³C NMR (DMSO-d₆): d

35.6, 39.4, 42.0, 55.6, 55.8, 111.6, 111.9, 119.5, 126.5, 129.3,

130.2, 131.3, 132.2, 131.9, 141.3, 147.9, 148.8, 165.1, 169.5. LC–

MS (ESI) purity 97%, m/z 421.3 [M+H]⁺, (method a). Anal

(C₁₉H₂₅ClN₄O₅S Â 2.0 H₂O) C, H, N.

4.3.33. 3-(2-(3,4-Dimethoxyphenylsulfonamido)acetamido)-

benzamidinium triﬂuoroacetate (33)

Yield 30%, mp 209–212 °C. ¹H NMR (DMSO-d₆): d 3.65 (d,

J = 6.3 Hz, 2H), 3.80 (s, 6H), 7.35 (d, J = 1.9 Hz, 1H), 7.39 (dd,

J = 8.2 Hz, J = 2.2 Hz, 1H), 7.42–7.44 (m, 1H), 7.53 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 7.75–7.78 (m, 1H), 7.93 (t, J = 6.0 Hz, 1H), 7.97

(dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 9.15, 9.29 (each s, 4H), 10.23 (s,

1H). APT ¹³C NMR (DMSO-d₆): d 46.1, 109.8, 111.3, 118.5, 120.5,

123.1, 124.1, 129.4, 129.7, 131.9, 139.2, 148.8, 152.2, 158.5 (q,

J = 32.8 Hz), 166.2, 167.2. LC–MS (ESI) purity 96%, m/z 393.4

[M+H]⁺, (method b). Anal (C₁₉H₂₁F₃N₄O₇S) C, H, N.

4.3.28. 3-(N-(4-(3,4-Dimethoxybenzylamino)-4-oxobutyl)-

sulfamoyl)benzamidinium chloride (28)

Yield 62%, mp 114–124 °C. ¹H NMR (DMSO-d₆): d 1.62 (tt,

J = 7.3 Hz, J = 7.3 Hz, 2H), 2.13 (t, J = 7.6 Hz, 2H), 2.75–2.78 (m,

2H), 3.70, 3.70 (each s, 6H), 4.13 (d, J = 5.7 Hz, 2H), 6.72 (dd,

J = 8.0 Hz, J = 1.9 Hz, 1H), 6.82 (d, J = 1.9 Hz, 1H), 6.84 (d,

J = 8.2 Hz, 1H), 7.82 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 7.92 (t,

J = 5.4 Hz, 1H), 8.05–8.07 (m, 1H), 8.08–8.10 (m, 1H), 8.25 (dd,

J = 1.9 Hz, J = 1.9 Hz, 1H), 8.30 (t, J = 5.7 Hz), 9.45, 9.66 (each s,

4H). APT ¹³C NMR (DMSO-d₆): d 25.6, 32.5, 41.9, 42.4, 55.6, 55.8,

111.5, 112.0, 119.4, 126.5, 129.2, 130.2, 131.2, 132.1, 132.2,

141.5, 147.9, 148.8, 165.1, 171.6. LC–MS (ESI) purity 97%, m/z

435.4 [M+H]⁺, (method a). Anal (C₂₀H₂₇ClN₄O₅S Â 1.8 H₂O) C, H, N.

4.3.34. 3-(2-(Naphthalene-2-sulfonamido)acetamido)benz-

amidinium triﬂuoroacetate (34)

Yield 44%, mp 249–253 °C. ¹H NMR (DMSO-d₆): d 3.74 (d,

J = 6.0 Hz, 2H), 7.39–7.41 (m, 1H), 7.49 (dd, J = 7.9 Hz, J = 7.9 Hz,

1H), 7.63–7.71 (m, 3H), 7.86 (dd, J = 8.9 Hz, J = 1.9 Hz, 1H), 7.92

(dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 8.02 (d, J = 8.2 Hz, 1H), 8.11–8.13

(m, 2H), 8.22 (t, J = 6.0 Hz, 1H), 8.45 (d, J = 1.6 Hz, 1H), 9.17, 9.27

(each s, 4H), 10.26 (s, 1H). APT ¹³C NMR (DMSO-d₆): d 46.0,

118.5, 122.5, 123.1, 124.0, 127.5, 127.7, 127.9, 128.9, 129.3,

129.4, 129.3, 131.8, 134.3, 137.6, 139.2, 158.6 (q, J = 31.0 Hz),

166.2, 167.1. LC–MS (ESI) purity 95%, m/z 383.2 [M+H]⁺, (method

b). Anal (C₂₁H₁₉F₃N₄O₅S Â 0.4 H₂O) C, H, N.

4.3.29. 3-(N-(2-Phenylamino)-2-oxoethyl)-N-sulfamoyl)-

benzamidinium triﬂuoroacetate (29)

Yield 46%, mp 87–99 °C. ¹H NMR (DMSO-d₆): d 3.75 (d, J = 6.0 Hz,

2H), 7.03 (tt, J = 8.5 Hz, J = 1.0 Hz), 7.27 (dd, J = 7.9 Hz, J = 7.9 Hz,

2H), 7.44 (dd, J = 8.5 Hz, J = 1.3 Hz, 2H), 7.83 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 8.01–8.03 (m, 1H), 8.14–8.17 (m, 1H), 8.21 (dd,

J = 1.6 Hz, J = 1.6 Hz, 1H), 8.28 (t, J = 6.0 Hz, 1H), 9.41, 9.49 (each s,

4H), 9.97 (s, 1H). APT ¹³C NMR (DMSO-d₆): d 45.9, 119.4, 123.6,

126.4, 128.9, 129.3, 130.2, 131.5, 132.1, 138.6, 141.6, 158.7 (q,

4.3.35. 3-(2-(Naphthalene-1-sulfonamido)acetamido)benz-

amidinium triﬂuoroacetate (35)

Yield 20%, mp 237–241 °C. ¹H NMR (DMSO-d₆): d 3.75 (d,

J = 6.3 Hz, 2H), 7.40–7.42 (m, 1H), 7.51 (dd, J = 7.9 Hz, J = 7.9 Hz,

6500

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

1H), 7.61–7.73 (m, 4H), 7.92 (dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 8.07 (dd,

J = 7.9 Hz, J = 0.6 Hz, 1H), 8.16 (dd, J = 7.6 Hz, J = 1.0 Hz, 1H), 8.20 (d,

J = 8.6 Hz, 1H), 8.49 (t, J = 6.3 Hz, 1H), 8.68 (dd, J = 1.6 Hz, J = 1.0 Hz,

1H), 9.21, 9.29 (each s, 4H), 10.23 (s, 1H). APT ¹³C NMR (DMSO-d₆):

d 45.8, 118.4, 123.1, 123.9, 124.6, 125.0, 127.0, 128.0, 128.4, 129.0,

127.7, 129.4, 129.7, 133.9, 134.0, 135.9, 139.1, 158.7 (q,

J = 31.0 Hz), 166.3, 167.3. LC–MS (ESI) purity 96%, m/z 383.2

[M+H]⁺, (method b). Anal (C₂₁H₁₉F₃N₄O₅S Â 0.5 H₂O) C, H, N.

2.85 (m, 2H), 7.39 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 7.54–7.56 (m,

1H), 7.79–7.82 (m, 1H), 7.94–7.98 (m, 5H), 8.13 (dd, J = 1.7 Hz,

J = 1.7 Hz, 1H), 9.44, 9.45 (each s, 4H), 10.08 (s, 1H). APT ¹³C NMR

(DMSO-d₆): d 25.1, 27.9, 33.3, 42.3, 80.9, 119.6, 123.3, 123.7,

126.9, 129.0, 129.4, 132.0, 132.1, 139.5, 145.1, 158.7 (q, J = 32.8),

165.0, 165.2, 170.9. LC–MS (ESI) purity 99%, m/z 461.4 [M+H]⁺,

(method c). Anal (C₂₄H₂₉F₃N₄O₇S Â 1.5 H₂O) C, H, N.

4.3.42. 3-(N-(2-(4-(tert-Butoxycarbonyl)phenylamino)-2-

oxoethyl)sulfamoyl)benzamidinium triﬂuoroacetate (42)

Yield 39%, mp 112–125 °C. ¹H NMR (DMSO-d₆): d 1.52 (s, 9H),

3.79 (d, J = 6.0 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.98 Hz,

2H), 7.83 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 8.01–8.03 (m, 1H), 8.14–

8.16 (m, 1H), 8.21 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 8.32 (t,

J = 6.0 Hz, 1H), 9.34, 9.48 (each s, 4H), 10.28 (s, 1H). APT ¹³C NMR

(DMSO-d₆): d 28.0, 45.9, 80.5, 118.6, 126.2, 126.4, 129.3, 130.2,

130.2, 131.5, 132.2, 141.6, 142.6, 158.6 (q, J = 32.5 Hz), 164.7,

164.9, 166.9. LC–MS (ESI) purity 96%, m/z 433.4 [M+H]⁺, (method

c). Anal (C₂₂H₂₅F₃N₄O₇S Â 1.9 H₂O) C, H, N.

4.3.36. 4-(N-(2-(4-(tert-Butoxycarbonyl)phenylamino)-2-

oxoethyl)sulfamoyl)benzamidinium triﬂuoroacetate (36)

Yield 23%, mp >250 °C. ¹H NMR (DMSO-d₆): d 1.52 (s, 9H), 3.80

(d, J = 6.0 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 8.9 Hz, 2H),

7.97, 8.02 (each d, J = 8.5 Hz, 4H), 8.43 (t, J = 6.0 Hz, 1H), 9.45, 9.46

(each s, 4H), 10.31 (s, 1H). APT ¹³C NMR (DMSO-d₆): d 28.0, 45.9,

80.5, 118.6, 126.1, 127.0, 129.2, 130.2, 132.0, 142.7, 145.3, 158.8

(q, J = 31.3 Hz), 164.7, 165.0, 166.9. LC–MS (ESI) purity 99%, m/z

433.3 [M+H]⁺, (method c). Anal (C₂₂H₂₅F₃N₄O₇S Â 0.5 H₂O) C, H, N.

4.3.37. 4-(N-(2-(3-(tert-Butoxycarbonyl)phenylamino)-2-

oxoethyl)sulfamoyl)benzamidinium triﬂuoroacetate (37)

Yield 32%, mp 212–215 °C. ¹H NMR (DMSO-d₆): d 1.53 (s, 9H),

3.78 (s, 2H), 7.40 (dd, J = 7.8 Hz, J = 7.8 Hz, 1H), 7.56–7.58 (m,

1H), 7.71–7.73 (m, 1H), 7.96, 8.03 (each d, J = 8.5 Hz, 4H), 8.04

(dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 8.41 (s, 1H), 9.33 (s, 4H), 10.18 (s,

1H). APT ¹³C NMR (DMSO-d₆): d 27.9, 46.2, 80.9, 119.8, 123.5,

124.1, 126.9, 128.5, 129.2, 132.0, 133.7, 138.9, 144.8, 164.8,

165.0, 166.9. LC–MS (ESI) purity 99%, m/z 433.4 [M+H]⁺, (method

c). Anal (C₂₂H₂₅F₃N₄O₇S Â 1.3 H₂O) C, H, N.

4.3.43. 3-(N-(2-(3-(tert-Butoxycarbonyl)phenylamino)-2-

oxoethyl)sulfamoyl)benzamidinium triﬂuoroacetate (43)

Yield 18%, mp 168–173 °C. ¹H NMR (DMSO-d₆): d 1.53 (s, 9H),

3.77 (s, 2H), 7.40 (dd, J = 7.8 Hz, J = 7.8 Hz, 1H), 7.56–7.59 (m,

1H), 7.72–7.75 (m, 1H), 7.83 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 8.01–

8.03 (m, 1H), 8.05 (dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 8.15–8.17 (m,

1H), 8.21 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 9.42 (s, 4H), 10.18 (s, 1H).

APT ¹³C NMR (DMSO-d₆): d 27.9, 45.9, 81.0, 119.9, 123.5, 124.2,

126.4, 129.2, 129.3, 130.2, 131.5, 132.2, 132.0, 138.9, 141.6, 158.6

(q, J = 31.0 Hz), 164.8, 165.0, 166.8. LC–MS (ESI) purity 99%, m/z

433.3 [M+H]⁺, (method c). Anal (C₂₂H₂₅F₃N₄O₇S) C, H, N.

4.3.38. 4-(N-(3-(4-(tert-Butoxycarbonyl)phenylamino)-3-

oxopropyl)sulfamoyl)benzamidinium triﬂuoroacetate (38)

Yield 19%, mp 223–225 °C. ¹H NMR (DMSO-d₆): d 1.52 (s, 9H),

2.57 (t, J = 7.0 Hz, 2H), 3.07 (t, J = 7.0 Hz, 2H), 7.66 (d, J = 8.9 Hz

2H), 7.82 (d, J = 8.8 Hz, 2H), 7.97, 8.00 (each d, J = 8.7 Hz, 4H),

9.38 (s, 4H), 10.28 (s, 1H). APT ¹³C NMR (DMSO-d₆): d 28.0, 36.8,

38.8, 80.4, 118.4, 125.8, 127.0, 129.4, 130.2, 132.2, 143.1, 144.7,

158.4 (q, J = 31.3 Hz), 164.7, 165.1, 169.3. LC–MS (ESI) purity 99%,

m/z 447.3 [M+H]⁺, (method c). Anal (C₂₃H₂₇F₃N₄O₇S) C, H, N.

4.3.44. 3-(N-(3-(4-(tert-Butoxycarbonyl)phenylamino)-3-

oxopropyl)sulfamoyl)benzamidinium triﬂuoroacetate (44)

Yield 70%, mp 127–135 °C. ¹H NMR (DMSO-d₆): d 1.52 (s, 9H),

2.56 (t, J = 7.0 Hz, 2H), 3.07–3.11 (m, 2H), 7.66 (d, J = 8.8 Hz, 2H),

7.83 (d, J = 8.8 Hz, 2H), 7.84 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 7.97 (t,

J = 6.0 Hz, 1H), 8.02–8.04 (m, 1H), 8.11–8.13 (m, 1H), 8.18 (dd,

J = 1.7 Hz, J = 1.7 Hz, 1H), 9.40, 9.50 (each s, 4H), 10.28 (s, 1H).

APT ¹³C NMR (DMSO-d₆): d 28.0, 36.8, 38.8, 80.4, 118.5, 125.8,

126.4, 129.6, 130.2, 130.3, 131.3, 132.2, 141.3, 143.2, 158.6 (q,

J = 31.9 Hz), 164.7, 165.1, 169.4. LC–MS (ESI) purity 99%, m/z

447.3 [M+H]⁺, (method c). Anal (C₂₃H₂₇F₃N₄O₇S Â 1.8 H₂O) C, H, N.

4.3.39. 4-(N-(3-(3-(tert-Butoxycarbonyl)phenylamino)-3-

oxopropyl)sulfamoyl)benzamidinium triﬂuoroacetate (39)

Yield 51%, mp 194–199 °C. ¹H NMR (DMSO-d₆): d 1.53 (s, 9H),

2.54 (t, J = 7.0 Hz, 2H), 3.05–3.09 (m, 2H), 7.39 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 7.55–7.57 (m, 1H), 7.78–7.80 (m, 1H), 7.97–8.05

(m, 5H), 8.13 (dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 9.30, 9.44 (each s,

4H), 10.16 (s, 1H). APT ¹³C NMR (DMSO-d₆): d 27.9, 36.7, 39.1,

80.9, 119.7, 123.4, 123.8, 126.9, 128.7, 129.0, 132.2, 134.6, 139.4,

143.8, 164.9, 164.9, 169.1. LC–MS (ESI) purity 96%, m/z 447.3

[M+H]⁺, (method c). Anal (C₂₃H₂₇F₃N₄O₇S Â 1.0 H₂O) C, H, N.

4.3.45. 3-(N-(3-(3-(tert-Butoxycarbonyl)phenylamino)-3-

oxopropyl)sulfamoyl)benzamidinium triﬂuoroacetate (45)

Yield 36%, mp 100–110 °C. ¹H NMR (DMSO-d₆): d 1.53 (s, 9H),

2.54 (t, J = 6.9 Hz, 2H), 3.07–3.11 (m, 2H), 7.40 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 7.55–7.57 (m, 1H), 7.79–7.81 (m, 1H), 7.85 (dd,

J = 7.9 Hz, J = 7.9 Hz, 1H), 7.96 (t, J = 5.7 Hz, 1H), 8.01–8.03 (m,

1H), 8.11–8.13 (m, 2H), 8.17 (dd, J = 1.7 Hz, J = 1.7 Hz, 1H), 9.19,

9.49 (each s, 4H), 10.15 (s, 1H). APT ¹³C NMR (DMSO-d₆): d 27.9,

36.8, 39.0, 80.9, 119.7, 123.4, 123.8, 125.7, 129.0, 129.9, 130.1,

131.3, 192.6, 132.3, 139.4, 141.1, 164.6, 164.9, 169.2. LC–MS (ESI)

4.3.40. 4-(N-(4-(4-(tert-Butoxycarbonyl)phenylamino)-4-

oxobutyl)sulfamoyl)benzamidinium triﬂuoroacetate (40)

Yield 14%, mp 122–137 °C. ¹H NMR (DMSO-d₆): d 1.52 (s, 9H),

1.71 (tt, J = 7.3 Hz, J = 7.3 Hz, 2H), 2.38 (t, J = 7.3 Hz, 2H), 2.81–

2.85 (m, 2H), 7.66 (d, J = 8.9 Hz, 2H), 7.82 (d, J = 8.9 Hz, 2H),

7.93–7.99 (m, 5H), 9.22, 9.43 (each s, 4H), 10.18 (s, 1H). APT ¹³C

NMR (DMSO-d₆): d 25.0, 28.0, 33.4, 42.3, 80.4, 118.4, 125.6,

126.8, 128.7, 130.2, 132.1, 143.2, 144.1, 164.7, 165.0, 171.2. LC–

MS (ESI) purity 95%, m/z 461.3 [M+H]⁺, (method c). Anal

(C₂₄H₂₉F₃N₄O₇S Â 1.1 H₂O) C, H, N.

purity

99%,

m/z

447.3

[M+H]⁺,

(method

c).

Anal

(C₂₃H₂₇F₃N₄O₇S Â 1.9 H₂O) C, H, N.

4.3.46. 3-(N-(4-(4-(tert-Butoxycarbonyl)phenylamino)-4-

oxobutyl)sulfamoyl)benzamidinium triﬂuoroacetate (46)

Yield 32%, mp 100–111 °C. ¹H NMR (DMSO-d₆): d 1.52 (s, 9H),

1.70 (tt, J = 7.3 Hz, J = 7.3 Hz, 2H), 2.37 (t, J = 7.3 Hz, 2H), 2.82–

3.86 (m, 2H), 7.66 (d, J = 8.9 Hz, 2H), 7.80–7.87 (m, 4H), 8.00–

8.02 (m, 1H), 8.09–8.11 (m, 1H), 8.16 (dd, J = 1.6 Hz, J = 1.6 Hz,

1H), 9.32, 9.49 (each s, 4H), 10.19 (s, 1H). APT ¹³C NMR (DMSO-

d₆): d 24.9, 28.0, 33.3, 42.2, 80.4, 118.4, 125.6, 126.3, 129.5,

130.2, 130.3, 131.2, 132.1, 141.5, 143.3, 158.5 (q, J = 32.8 Hz),

4.3.41. 4-(N-(4-(3-(tert-Butoxycarbonyl)phenylamino)-4-

oxobutyl)sulfamoyl)benzamidinium triﬂuoroacetate (41)

Yield 70%, mp 123–138 °C. ¹H NMR (DMSO-d₆): d 1.53 (s, 9H),

1.72 (tt, J = 7.3 Hz, J = 7.3 Hz, 2H), 2.36 (t, J = 7.3 Hz, 2H), 2.82–

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

6501

164.7, 165.0, 171.2. LC–MS (ESI) purity 98%, m/z 461.4 [M+H]⁺,

(method c). Anal (C₂₄H₂₉F₃N₄O₇S Â 1.6 H₂O) C, H, N.

2H), 7.66 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H), 7.95 (t,

J = 5.7 Hz, 1H), 7.98 (s, 4H), 9.45 (s, 4H), 10.19 (s, 1H), 12.64 (s,

1H). APT ¹³C NMR (DMSO-d₆): d 25.1, 33.4, 42.3, 118.4, 125.1,

126.9, 129.5, 130.5, 132.1, 143.4, 145.1, 165.2, 167.0, 171.2. LC–

MS (ESI) purity 95%, m/z 405.4 [M+H]⁺, (method d). Anal

(C₂₀H₂₁F₃N₄O₇S Â 1.1 H₂O) C, H, N.

4.3.47. 3-(N-(4-(3-(tert-Butoxycarbonyl)phenylamino)-4-

oxobutyl)sulfamoyl)benzamidinium triﬂuoroacetate (47)

Yield 47%, mp 91–100 °C. ¹H NMR (DMSO-d₆): d 1.53 (s, 9H),

1.71 (tt, J = 7.3 Hz, J = 7.3 Hz, 2H), 2.35 (t, J = 7.3 Hz, 2H), 2.82–

2.86 (m, 2H), 7.39 (dd, J = 8.1 Hz, J = 8.1 Hz, 1H), 7.54–7.56 (m,

1H), 7.80–7.82 (m, 1H), 7.84 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 7.87

(t, J = 6.0 Hz, 1H), 8.01–8.03 (m, 1H), 8.10–8.12 (m, 2H), 8.16 (dd,

J = 1.6 Hz, J = 1.6 Hz, 1H), 9.39, 9.49 (each s, 4H), 10.08 (s, 1H).

APT ¹³C NMR (DMSO-d₆): d 25.1, 27.9, 33.3, 42.2, 80.9, 119.6,

123.3, 123.7, 126.3, 129.0, 129.6, 130.3 131.2, 132.1, 131.9, 139.6,

141.5, 158.6 (q, J = 32.8 Hz), 165.0, 165.1, 171.0. LC–MS (ESI) purity

99%, m/z 461.3 [M+H]⁺, (method c). Anal (C₂₄H₂₉F₃N₄O₇S Â 1.7

H₂O) C, H, N.

4.3.53. 4-(N-(4-(3-Carboxylphenylamino)-4-oxobutyl)-

sulfamoyl)benzamidinium triﬂuoroacetate (53)

Yield >99%, mp 167–171 °C. ¹H NMR (DMSO-d₆): d 1.72 (tt,

J = 7.3 Hz, J = 7.3 Hz, 2H), 2.36 (t, J = 7.3 Hz, 2H), 2.82–2.86 (m,

2H), 7.39 (dd, J = 8.2 Hz, J = 8.2 Hz, 1H), 7.58–7.60 (m, 1H), 7.75–

7.78 (m, 1H), 7.95 (t, J = 6.0 Hz, 1H), 7.97, 7.98 (each s, 4H), 8.20

(dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 9.37, 9.44 (each s, 4H), 10.06 (s, 1H),

12.90 (s, 1H). APT ¹³C NMR (DMSO-d₆): d 25.1, 33.3, 42.3, 119.9,

123.3, 124.0, 126.9, 129.0, 129.5, 131.4, 132.1, 139.5, 145.1, 158.6

(q, J = 31.0 Hz), 165.1, 167.3, 170.9. LC–MS (ESI) purity 96%, m/z

405.4 [M+H]⁺, (method d). Anal (C₂₀H₂₁F₃N₄O₇S Â 1.0 H₂O) C, H, N.

4.3.48. 4-(N-(2-(4-Carboxylphenylamino)-2-oxoethyl)-

sulfamoyl)benzamidinium triﬂuoroacetate (48)

Yield >99%, mp >250 °C. ¹H NMR (DMSO-d₆):

3.81 (d,

4.3.54. 3-(N-(2-(4-Carboxylphenylamino)-2-oxoethyl)-

J = 6.0 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 8.9 Hz, 2H),

7.96, 8.02 (each d, J = 8.7 Hz, 4H), 8.43 (t, J = 6.1 Hz, 1H), 9.35,

9.44 (each s, 4H), 10.29 (s, 1H), 12.66 (s, 1H). APT ¹³C NMR

(DMSO-d₆): d 45.9, 118.7, 125.6, 127.0, 129.2, 130.5, 131.9, 142.6,

145.4, 165.0, 166.9, 167.0. LC–MS (ESI) purity 98%, m/z 377.5

[M+H]⁺, (method d). Anal (C₁₈H₁₇F₃N₄O₇S Â 1.1 H₂O) C, H, N.

sulfamoyl)benzamidinium triﬂuoroacetate (54)

Yield >99%, mp 140–151 °C. ¹H NMR (DMSO-d₆): d 3.80 (d,

J = 6.0 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 7.84 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 7.87 (d, J = 8.9 Hz, 2H), 8.01–8.03 (m, 1H), 8.14–

8.16 (m, 1H), 8.21 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 8.24 (t,

J = 6.1 Hz, 1H), 9.39, 9.48 (each s, 4H), 10.28 (s, 1H), 12.72 (s, 1H).

APT ¹³C NMR (DMSO-d₆): d 46.0, 118.6, 125.6, 126.3, 129.3,

130.2, 131.5, 132.2, 130.5, 141.6, 142.6, 158.7 (q, J = 31.9 Hz,

165.0, 166.9, 167.0. LC–MS (ESI) purity 95%, m/z 377.4 [M+H]⁺,

(method d). Anal (C₁₈H₁₇F₃N₄O₇S Â 2.0 H₂O) C, H, N.

4.3.49. 4-(N-(2-(3-Carboxylphenylamino)-2-oxoethyl)-

sulfamoyl)benzamidinium triﬂuoroacetate (49)

Yield >99%, mp 236–240 °C. ¹H NMR (DMSO-d₆): d 3.78 (d,

J = 6.0 Hz, 2H), 7.40 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 7.60–7.62 (m,

1H), 7.69–7.72 (m, 1H), 7.97, 8.03 (each d, J = 8.8 Hz, 4H), 8.12

(dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 8.42 (t, J = 6.0 Hz, 1H), 9.36, 9.45

(each s, 4H), 10.18 (s, 1H), 12.85 (s, 1H). APT ¹³C NMR (DMSO-

d₆): d 45.9, 120.2, 123.5, 124.5, 127.0, 129.1, 129.2, 131.5, 131.9,

138.9, 145.4, 158.7 (q, J = 32.8 Hz), 165.0, 166.6, 167.2. LC–MS

(ESI) purity 94%, m/z 377.4 [M+H]⁺, (method d). Anal

(C₁₈H₁₇F₃N₄O₇S Â 1.4 H₂O) C, H, N.

4.3.55. 3-(N-(2-(3-Carboxylphenylamino)-2-oxoethyl)-

sulfamoyl)benzamidinium triﬂuoroacetate (55)

Yield >99%, mp >250 °C. ¹H NMR (DMSO-d₆):

d 3.77 (d,

J = 6.0 Hz, 2H), 7.41 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 7.61–7.63 (m,

1H), 7.70–7.73 (m, 1H), 7.83 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 8.01–

8.03 (m, 1H), 8.11 (dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 8.15–8.17 (m,

1H), 8.21 (dd, J = 1.6 Hz, J = 1.6 Hz, 2H), 8.31 (t, J = 6.2 Hz, 1H),

9.37, 9.49 (each s, 4H), 10.16 (s, 1H), 12.96 (s, 1H). APT ¹³C NMR

(DMSO-d₆): d 45.9, 120.2, 123.5, 124.4, 126.3, 129.2, 129.3, 130.2,

131.5, 132.2, 131.5, 138.8, 141.6, 158.7 (q, J = 31.0 Hz), 165.0,

166.7, 167.2. LC–MS (ESI) purity 97%, m/z 377.3 [M+H]⁺, (method

d). Anal (C₁₈H₁₇F₃N₄O₇S Â 0.5 H₂O) C, H, N.

4.3.50. 4-(N-(3-(4-Carboxylphenylamino)-3-oxopropyl)-

sulfamoyl)benzamidinium triﬂuoroacetate (50)

Yield >99%, mp 146–152 °C. ¹H NMR (DMSO-d₆): d 2.57 (t,

J = 7.0 Hz, 2H), 3.07–3.10 (m, 2H), 7.66 (d, J = 8.9 Hz, 2H), 7.86 (d,

J = 8.9 Hz, 2H), 7.97, 8.00 (each d, J = 8.7 Hz, 4H), 8.05 (t,

J = 5.7 Hz, 1H), 9.37, 9.45 (each s, 4H), 10.27 (s, 1H), 12.63 (s, 1H).

APT ¹³C NMR (DMSO-d₆): d 36.8, 38.9, 118.5, 125.2, 127.0, 129.4,

130.5, 132.1, 143.1, 144.7, 158.5 (q, J = 31.9 Hz), 165.1, 167.0,

169.3. LC–MS (ESI) purity 99%, m/z 391.3 [M+H]⁺, (method d). Anal

(C₁₉H₁₉F₃N₄O₇S Â 1.8 H₂O) C, H, N.

4.3.56. 3-(N-(3-(4-Carboxylphenylamino)-3-oxopropyl)-

sulfamoyl)benzamidinium triﬂuoroacetate (56)

Yield >99%, mp 224–229 °C. ¹H NMR (DMSO-d₆): d 2.56 (t,

J = 6.9 Hz, 2H), 3.07–3.11 (m, 2H), 7.66 (d, J = 8.9 Hz, 2H), 7.84

(dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.97 (t,

J = 6.0 Hz, 1H), 8.01–8.03 (m, 1H), 8.11–8.13 (m, 1H), 8.18 (dd,

J = 1.9 Hz, J = 1.9 Hz), 9.43, 9.49 (each s, 4H), 10.27 (s, 1H), 12.66

(s, 1H). APT ¹³C NMR (DMSO-d₆): d 36.9, 38.8, 118.5, 125.3,

126.4, 129.6, 130.3, 131.3, 132.2, 130.5, 141.3, 143.2, 158.8 (q,

J = 31.0 Hz), 165.1, 167.0, 169.4. LC–MS (ESI) purity 99%, m/z

391.4 [M+H]⁺, (method d). Anal (C₁₉H₁₉F₃N₄O₇S Â 1.1 H₂O) C, H, N.

4.3.51. 4-(N-(3-(3-Carboxylphenylamino)-3-oxopropyl)-

sulfamoyl)benzamidinium triﬂuoroacetate (51)

Yield >99%, mp 122–132 °C. ¹H NMR (DMSO-d₆): d 2.55 (t,

J = 7.0 Hz, 2H), 3.06–3.10 (m, 2H), 7.40 (dd, J = 7.9 Hz, J = 7.9 Hz,

1H), 7.59–7.61 (m, 1H), 7.75–7.77 (m, 1H), 7.99, 8.00 (each s,

4H), 8.04 (t, J = 5.7 Hz, 1H), 8.21 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H),

9.44, 9.45 (each s, 4H), 10.16 (s, 1H), 12.81 (s, 1H). APT ¹³C NMR

(DMSO-d₆): d 36.8, 39.0, 120.1, 123.3, 124.1, 127.0, 129.1, 129.4,

131.4, 132.2, 139.4, 144.8, 158.7 (q, J = 31.0 Hz), 165.2, 167.3,

169.0. LC–MS (ESI) purity 97%, m/z 391.5 [M+H]⁺, (method d). Anal

(C₁₉H₁₉F₃N₄O₇S Â 1.0 H₂O) C, H, N.

4.3.57. 3-(N-(3-(3-Carboxylphenylamino)-3-oxopropyl)-

sulfamoyl)benzamidinium triﬂuoroacetate (57)

Yield >99%, mp 113–125 °C. ¹H NMR (DMSO-d₆): d 2.53 (t,

J = 6.9 Hz, 2H), 3.07–3.11 (m, 2H), 7.40 (dd, J = 8.2 Hz, J = 8.2 Hz,

1H), 7.59–7.61 (m, 1H), 7.76–7.78 (m, 1H), 7.85 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 7.96 (t, J = 6.0 Hz, 1H), 8.01–8.03 (m, 1H), 8.11–

8.13 (m, 1H), 8.16–8.20 (m, 2H), 9.35, 9.49 (each s, 4H), 10.15 (s,

1H), 12.89 (s, 1H). APT ¹³C NMR (DMSO-d₆): d 36.8, 38.9, 120.1,

123.3, 124.1, 126.4, 129.1, 129.6, 130.3, 131.3, 132.2, 131.4,

139.4, 141.3, 158.5 (q, J = 31.3 Hz), 165.1, 167.3, 169.1. LC–MS

4.3.52. 4-(N-(4-(4-Carboxylphenylamino)-4-oxobutyl)-

sulfamoyl)benzamidinium triﬂuoroacetate (52)

Yield >99%, mp 156–162 °C. ¹H NMR (DMSO-d₆): d 1.72 (tt,

J = 7.0 Hz, J = 7.0 Hz, 2H), 2.38 (t, J = 7.3 Hz, 2H), 2.82–2.86 (m,

6502

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

(ESI) purity 98%, m/z 391.3 [M+H]⁺, (method d). Anal

(C₁₉H₁₉F₃N₄O₇S Â 1.6 H₂O) C, H, N.

4.3.63. 3-(N-(2-(4-Amidiniobenzylamino)-2-oxoethyl)-

sulfamoyl)benzamidinium (bis)triﬂuoroacetate (63)

Yield 49%, mp 232–235 °C. ¹H NMR (DMSO-d₆): d 3.56 (d,

J = 6.3 Hz, 2H), 4.33 (d, J = 6.0 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.76

(d, J = 8.2 Hz, 2H), 7.84 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 8.03–8.05 (m,

1H), 8.12–8.14 (m, 1H), 8.19 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 8.31 (t,

J = 6.3 Hz, 1H), 8.59 (t, J = 6.0 Hz, 1H), 9.26, 9.50 (each s, 8H). APT

¹³C NMR (DMSO-d₆): d 41.9, 45.3, 117.2 (q, J = 297.0 Hz), 126.4,

126.7, 127.5, 128.2, 129.4, 130.3, 131.4, 132.2, 141.2, 145.7, 158.8

(q, J = 31.0 Hz), 165.1, 165.6, 168.1. LC–MS (ESI) purity 97%, m/z

389.5 [M+H]⁺, (method d). Anal (C₂₁H₂₂F₆N₆O₇S Â 0.5 H₂O) C, H, N.

4.3.58. 3-(N-(4-(4-Carboxylphenylamino)-4-oxobutyl)-

sulfamoyl)benzamidinium triﬂuoroacetate (58)

Yield >99%, mp 152–155 °C. ¹H NMR (DMSO-d₆): d 1.71 (tt,

J = 7.0 Hz, J = 7.0 Hz, 2H), 2.38 (t, J = 7.3 Hz, 2H), 2.83–2.86 (m,

2H), 7.66 (d, J = 8.8 Hz, 2H), 7.82–7.87 (m, 4H), 8.00–8.02 (m,

1H), 8.09–8.11 (m, 1H), 8.16 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 9.36,

9.49 (each s, 4H), 10.19 (s, 1H), 12.66 (s, 1H). APT ¹³C NMR

(DMSO-d₆): d 25.0, 33.4, 42.2, 118.4, 125.1, 126.3, 129.5, 130.3,

131.2, 132.1, 130.5, 141.5, 143.3, 158.7 (q, J = 31.9 Hz), 165.1,

167.0, 171.2. LC–MS (ESI) purity 95%, m/z 405.5 [M+H]⁺, (method

d). Anal (C₂₀H₂₁F₃N₄O₇S Â 0.6 H₂O) C, H, N.

4.3.64. 3-(N-(3-(4-Amidiniobenzylamino)-3-oxopropyl)-

sulfamoyl)benzamidinium bis(triﬂuoroacetate) (64)

Yield 71%, mp 216–220 °C. ¹H NMR (DMSO-d₆): d 2.38 (t,

J = 7.3 Hz, 2H), 3.00–3.04 (m, 2H), 4.33 (d, J = 6.0 Hz, 2H), 7.45 (d,

J = 8.5 Hz, 2H), 7.75 (d, J = 8.2 Hz, 2H), 7.84 (dd, J = 7.9 Hz,

J = 7.9 Hz, 1H), 7.91 (t, J = 5.7 Hz, 1H), 8.02–8.04 (m, 1H), 8.10–

8.12 (m, 1H), 8.17 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 8.56 (t,

J = 6.0 Hz, 1H), 9.24, 9.26, 9.48, 9.51 (each s, 8H). APT ¹³C NMR

(DMSO-d₆): d 35.6, 41.9, 126.3, 126.6, 127.6, 128.2, 129.6, 130.3,

131.2, 132.1, 141.3, 146.0, 158.8 (q, J = 31.0 Hz), 165.1, 165.6,

170.1. LC–MS (ESI) purity 99%, m/z 403.5 [M+H]⁺, (method d). Anal

(C₂₂H₂₄F₆N₆O₇S Â 1.0 H₂O) C, H, N.

4.3.59. 3-(N-(4-(3-Carboxylphenylamino)-4-oxobutyl)-

sulfamoyl)benzamidinium triﬂuoroacetate (59)

Yield >99%, mp 92–111 °C. ¹H NMR (DMSO-d₆): d 1.71 (tt,

J = 7.2 Hz, J = 7.2 Hz, 2H), 2.35 (t, J = 7.3 Hz, J = 7.3 Hz, 2H), 2.82–

2.86 (m, 2H), 7.39 (dd, J = 8.2 Hz, J = 8.2 Hz, 1H), 7.58–7.60 (m,

1H), 7.76–7.78 (m, 1H), 7.84 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 7.87

(t, J = 6.0 Hz, 1H), 8.00–8.02 (m, 1H), 8.10–8.12 (m, 1H), 8.16 (dd,

J = 1.6 Hz, J = 1.6 Hz, 1H), 8.20 (dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 9.35,

9.49 (each s, 4H), 10.06 (s, 1H), 12.81 (s, 1H). APT ¹³C NMR

(DMSO-d₆): d 25.1, 32.2, 42.2, 119.9, 123.2, 123.9, 126.3, 129.0,

129.5, 130.3, 131.1, 132.1, 131.4, 139.1, 141.5, 158.5 (q,

J = 33.7 Hz, 165.0, 167.2, 170.9. LC–MS (ESI) purity 96%, m/z 405.4

[M+H]⁺, (method d). Anal (C₂₀H₂₁F₃N₄O₇S Â 1.0 H₂O) C, H, N.

4.3.65. 3-(N-(4-(4-Amidiniobenzylamino)-4-oxobutyl)-

sulfamoyl)benzamidinium bis(triﬂuoroacetate) (65)

Yield 39%, mp 212–218 °C. ¹H NMR (DMSO-d₆): d 1.63 (tt,

J = 7.3 Hz, J = 7.3 Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 2.78–2.82 (m,

2H), 4.32 (d, J = 6.0 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H), 7.76 (d,

J = 8.5 Hz, 2H), 7.82–7.86 (m, 2H), 8.02–8.04 (m, 1H), 8.08–8.10

(m, 1H), 8.16 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 8.47 (t, J = 6.0 Hz, 1H),

9.24, 9.26, 9.49, 9.51 (each s, 8H). APT ¹³C NMR (DMSO-d₆): d

25.4, 32.3, 41.9, 42.4, 117.3 (q, J = 297.9 Hz), 126.3, 126.6, 127.5,

128.3, 129.6, 130.3, 131.2, 132.1, 141.5, 146.2, 158.8 (q,

J = 31.0 Hz), 165.2, 165.6, 171.9. LC–MS (ESI) purity 98%, m/z

417.3 [M+H]⁺, (method d). Anal (C₂₃H₂₆F₆N₆O₇S Â 2.0 H₂O) C, H, N.

4.3.60. 4-(N-(2-(4-Amidiniobenzylamino)-2-oxoethyl)-

sulfamoyl)benzamidinium bis(triﬂuoroacetate) (60)

Yield 26%, mp 211–216 °C. ¹H NMR (DMSO-d₆): d 3.55 (d,

J = 6.0 Hz, 2H), 4.34 (d, J = 6.0 Hz, 2H), 7.45 (d, J = 8.2 Hz, 2H),

7.76 (d, J = 8.5 Hz, 2H), 7.98, 8.01 (each d, J = 8.5 Hz, 4H), 8.40 (t,

J = 6.0 Hz, 1H), 8.61 (t, J = 6.2 Hz, 1H), 9.22, 9.25, 9.47, 9.49 (each

s, 8H). APT ¹³C NMR (DMSO-d₆): d 41.9, 45.4, 126.7, 127.0, 127.6,

128.2, 129.3, 132.1, 144.9, 145.7, 158.7 (q, J = 31.0 Hz), 165.1,

165.6, 168.0. LC–MS (ESI) purity 99%, m/z 389.4 [M+H]⁺, (method

d). Anal (C₂₁H₂₂F₆N₆O₇S Â 0.7 H₂O) C, H, N.

4.3.66. (S)-4-(N-(1-(4-Amidiniobenzylamino)-1-oxo-3-

phenylpropan-2-yl)sulfamoyl)benzamidinium bis(triﬂuoro-

acetate) (66)

Yield 10%, mp 256–259 °C. ¹H NMR (DMSO-d₆): d 2.66–2.72,

2.86–2.89 (each m, 2H), 4.08–4.14 (m, 2H), 4.24 (dd, J = 16.1 Hz,

J = 6.6 Hz, 1H), 7.09–7.11 (m, 2H), 7.17–7.18 (m, 3H), 7.20 (d,

J = 8.2 Hz, 2H), 7.69 (d, J = 8.2 Hz, 2H), 7.75, 7.84 (each d,

J = 8.8 Hz, 4H), 8.56 (d, J = 8.9 Hz, 1H), 8.63 (t, J = 5.7 Hz, 1H),

9.05, 9.23, 9.32, 9.43 (each s, 8H). APT ¹³C NMR (DMSO-d₆): d

38.5, 41.7, 58.2, 117.4 (q, J = 297.9 Hz), 126.6, 126.7, 126.7, 127.3,

128.2, 128.4, 128.9, 129.4, 131.5, 136.8, 145.5, 145.9, 158.7 (q,

J = 31.0 Hz), 164.9, 165.5, 170.4. LC–MS (ESI) purity 99%, m/z

479.3 [M+H]⁺, (method d). Anal (C₂₈H₂₈F₆N₆O₇S Â 2.9 H₂O) C, H, N.

4.3.61. 4-(N-(3-(4-Amidiniobenzylamino)-3-oxopropyl)-

sulfamoyl)benzamidinium bis(triﬂuoroacetate) (61)

Yield 28%, mp 210–211 °C. ¹H NMR (DMSO-d₆): d 2.39 (t,

J = 7.3 Hz, 2H), 2.98–3.02 (m, 2H), 4.33 (d, J = 6.0 Hz, 2H), 7.45 (d,

J = 8.5 Hz, 2H), 7.76 (d, J = 8.2 Hz, 2H), 7.99 (s, 5H), 8.56 (t,

J = 6.0 Hz, 1H), 9.26, 9.47, 9.53 (each s, 8H). APT ¹³C NMR (DMSO-

d₆): d 35.7, 39.3, 41.9, 126.7, 127.0, 127.6, 128.2, 129.4, 132.2,

144.7, 146.0, 158.9 (q, J = 31.9 Hz), 165.2, 165.6, 170.0. LC–MS

(ESI) purity 99%, m/z 403.5 [M+H]⁺, (method d). Anal

(C₂₂H₂₄F₆N₆O₇S Â 0.3 H₂O) C, H, N.

4.3.67. (R)-4-(N-(1-(4-Amidiniobenzylamino)-1-oxo-3-

phenylpropan-2-yl)sulfamoyl)benzamidinium bis(triﬂuoro-

acetate) (67)

4.3.62. 4-(N-(4-(4-Amidiniobenzylamino)-4-oxobutyl)-

sulfamoyl)benzamidinium bis(triﬂuoroacetate) (62)

Yield 22%, mp 255–257 °C. ¹H NMR (DMSO-d₆): d 2.68, 2.84

(each dd, J = 13.6 Hz, J = 6.6 Hz, 2H), 4.06–4.13 (m, 2H), 4.24 (dd,

J = 16.1 Hz, J = 6.3 Hz, 1H), 7.09–7.11 (m, 2H), 7.16–7.18 (m, 3H),

7.20 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 8.6 Hz, 2H), 7.75, 7.84 (each d,

J = 8.8 Hz, 4H), 8.56 (d, J = 8.8 Hz, 1H), 8.64 (t, J = 6.0 Hz, 1H),

9.02, 9.23, 9.29, 9.42 (each s, 8H). APT ¹³C NMR (DMSO-d₆): d

38.4, 41.7, 58.2, 126.5, 126.7, 126.7, 127.3, 128.1, 128.4, 128.9,

129.4, 131.5, 136.8, 145.5, 145.9, 158.7 (q, J = 31.9 Hz), 164.9,

165.5, 170.4. LC–MS (ESI) purity 99%, m/z 479.3 [M+H]⁺, (method

d). Anal (C₂₈H₂₈F₆N₆O₇S Â 2.2 H₂O) C, H, N.

Yield 10%, mp 238–239 °C. ¹H NMR (DMSO-d₆): d 1.65 (tt,

J = 7.3 Hz, J = 7.3 Hz, 2H), 2.19 (t, J = 7.3 Hz, 2H), 2.78–2.81 (m,

2H), 4.32 (d, J = 5.7 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.76 (d,

J = 8.2 Hz, 2H), 7.93 (t, J = 5.7 Hz, 1H), 7.98 (s, 4H), 8.46 (t,

J = 6.0 Hz, 1H), 9.25, 9.47, 9.53 (each s, 8H). APT ¹³C NMR (DMSO-

d₆): d 25.5, 32.3, 41.9, 42.4, 126.6, 126.9, 127.5, 128.2, 129.4,

132.1, 145.1, 146.2, 158.8 (q, J = 31.9 Hz), 165.2, 165.6, 171.8. LC–

MS (ESI) purity 98%, m/z 417.4 [M+H]⁺, (method d). Anal

(C₂₃H₂₆F₆N₆O₇S Â 0.4 H₂O) C, H, N.

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

6503

4.3.68. (S)-4-(N-(1-(4-Amidiniobenzylamino)-3-benzyloxy-1-

oxopropan-2-yl)sulfamoyl)benzamidinium

bis(triﬂuoroacetate) (68)

2H), 7.27–7.35 (m, 3H), 7.39 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 8.5 Hz,

2H), 7.77 (dd, J = 7.9 Hz, J = 7.9 Hz, 1H), 8.02–8.04 (m, 1H), 8.14–

8.16 (m, 1H), 8.24 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 8.48 (d,

J = 8.5 Hz, 1H), 8.66 (t, J = 6.3 Hz, 1H), 9.50, 9.54 (each s, 8H). APT

¹³C NMR (DMSO-d₆): d 41.9, 56.3, 69.9, 72.2, 126.5, 126.6, 127.3,

127.6, 128.1, 128.3, 129.1, 130.1, 131.5, 132.1, 137.9, 141.9,

145.5, 145.8, 158.7 (q, J = 31.0 Hz), 165.0, 165.6, 169.0. LC–MS

(ESI) purity 99%, m/z 509.3 [M+H]⁺, (method d). Anal

(C₂₉H₃₀F₆N₆O₈S Â 2.4 H₂O) C, H, N.

Yield 14%, mp 243–244 °C. ¹H NMR (DMSO-d₆): d 3.46, 3.50

(each dd, J = 9.8 Hz, 6.0 Hz, 2H), 4.10 (dd, J = 8.2 Hz, J = 6.3 Hz,

1H), 4.23, 4.33 (each dd, J = 15.1 Hz, J = 6.0 Hz, 2H), 4.33–4.35 (m,

2H), 7.19–7.21 (m, 2H), 7.25–7.32 (m, 3H), 7.40 (d, J = 8.5 Hz,

2H), 7.69 (d, J = 8.2 Hz, 2H), 7.92, 8.00 (each d, J = 8.5 Hz. 4H),

8.55 (d, J = 8.2 Hz, 1H), 8.73 (t, J = 6.0 Hz, 1H), 9.21, 9.23, 9.46,

9.50 (each s, 8H). APT ¹³C NMR (DMSO-d₆): d 41.9, 56.4, 69.9,

72.2, 117.3 (q, J = 197.9 Hz), 126.6, 127.0, 127.3, 127.6, 128.1,

128.3, 129.0, 131.7, 137.9, 145.5, 145.8, 158.7 (q, J = 31.0 Hz),

164.9, 165.6, 169.0. LC–MS (ESI) purity 99%, m/z 509.3 [M+H]⁺,

(method d). Anal (C₂₉H₃₀F₆N₆O₈S Â 2.5 H₂O) C, H, N.

4.3.73. (R)-3-(N-(1-(4-Amidiniobenzylamino)-3-benzyloxy-1-

oxopropan-2-yl)sulfamoyl)benzamidinium bis(triﬂuoro-

acetate) (73)

Yield 14%, mp 204–205 °C. ¹H NMR (DMSO-d₆): d 3.44–3.51 (m,

2H), 4.07 (t, J = 5.4 Hz, 1H), 4.17, 4.29 (dd, J = 16.1 Hz, J = 5.7 Hz,

2H), 4.35 (d, J = 7.6 Hz, 2H), 7.20–7.22 (m, 2H), 7.25–7.33 (m,

3H), 7.38 (d, J = 8.6 Hz, 2H), 7.68 (d, J = 8.2 Hz, 2H), 7.75 (dd,

J = 7.9 Hz, J = 7.9 Hz, 1H), 7.99–8.01 (m, 1H), 8.12–8.15 (m, 1H),

8.21 (dd, J = 1.9 Hz, J = 1.9 Hz, 1H), 8.46 (s, 1H), 8.63 (t, J = 6.3 Hz,

1H), 9.29 (s, 8H). APT ¹³C NMR (DMSO-d₆): d 41.9, 56.3, 69.9,

72.2, 126.5, 126.6, 127.3, 127.6, 128.1, 128.3, 129.1, 130.1, 131.5,

132.1, 137.9, 141.9, 145.5, 145.8, 158.7 (q, J = 31.0 Hz), 165.0,

165.6, 169.0. LC–MS (ESI) purity 99%, m/z 509.4 [M+H]⁺, (method

d). Anal (C₂₉H₃₀F₆N₆O₈S Â 2.7 H₂O) C, H, N.

4.3.69. (R)-4-(N-(1-(4-Amidiniobenzylamino)-3-benzyloxy-1-

oxopropan-2-yl)sulfamoyl)benzamidinium bis(triﬂuoro-

acetate) (69)

Yield 9%, mp 245–249 °C. ¹H NMR (DMSO-d₆): d 3.46, 3.48 (each

dd, J = 10.1 Hz, 6.3 Hz, 2H), 4.10 (dd, 8.2 Hz, J = 6.3 Hz, 1H), 4.23,

4.32 (each dd, J = 16.1 Hz, J = 5.7 Hz, 2H), 4.33–4.35 (m, 2H), 7.19–

7.21 (m, 2H), 7.26–7.32 (m, 3H), 7.40 (d, J = 8.5 Hz, 2H), 7.69 (d,

J = 8.5 Hz, 2H), 7.92, 7.99 (each d, J = 8.5 Hz. 4H), 8.55 (d, J = 8.2 Hz,

1H), 8.72 (t, J = 6.0 Hz, 1H), 9.20, 9.23, 9.46, 9.49 (each s, 8H). APT

¹³C NMR (DMSO-d₆): d 41.9, 56.4 69.9, 72.2, 126.6, 127.0, 127.3,

127.6, 128.1, 128.3, 129.0, 131.7, 137.9, 145.5, 145.8, 158.7 (q,

J = 31.0 Hz), 164.9, 165.6, 169.0. LC–MS (ESI) purity 99%, m/z 509.4

[M+H]⁺, (method d). Anal (C₂₉H₃₀F₆N₆O₈S Â 2.3 H₂O) C, H, N.

4.4. Enzyme kinetics

4.4.1. Assay conditions

The activity of bovine trypsin and of matriptase-2 in the condi-

tioned medium of transfected HEK cells (HEK-MT2 cells) was

determined by monitoring the release of 7-amino-4-methylcoum-

arin from the ﬂuorogenic substrate Boc-Gln-Ala-Arg-NH-Mec.

Cbz-Gly-Gly-Arg-NH-Mec was used for the determination of the

activity of human thrombin. Reactions were monitored with an

excitation wavelength of 340 nm and emission wavelength of

460 nm. Inhibition assays were performed in duplicate measure-

ments with ﬁve different inhibitor concentrations. Steady-state

velocities were plotted against inhibitor concentrations to obtain

4.3.70. (S)-3-(N-(1-(4-Amidiniobenzylamino)-1-oxo-3-

phenylpropan-2-yl)sulfamoyl)benzamidinium bis(triﬂuoro-

acetate) (70)

Yield 16%, mp 225–231 °C. ¹H NMR (DMSO-d₆): d 2.70, 2.89

(each dd, J = 13.6 Hz, J = 6.3 Hz, 2H), 4.00–4.07 (m, 2H), 4.20 (dd,

J = 16.1 Hz, J = 6.3 Hz, 1H), 7.09–7.10 (m, 2H), 7.15–7.17 (m, 3H),

7.18 (d, J = 8.6 Hz, 2H), 7.66 (dd, J = 7.9 H, J = 7.9 Hz, 1H), 7.70 (d,

J = 8.5 Hz, 2H), 7.84–7.86 (m, 1H), 7.94–7.96 (m, 1H), 8.00 (dd,

J = 1.6 Hz, J = 1.6 Hz, 1H), 8.48 (d, J = 9.2 Hz, 1H), 8.57 (t,

J = 5.7 Hz, 1H), 9.07, 9.24, 9.31, 9.43 (each s, 8H). APT ¹³C NMR

(DMSO-d₆): d 38.4, 41.7, 58.1, 117.2 (q, J = 279.0 Hz), 126.2,

126.6, 126.7, 127.2, 128.1, 128.2, 128.9, 129.3, 129.9, 131.2,

131.8, 136.8, 141.9, 145.3, 158.8 (q, J = 31.0 Hz), 164.9, 165.5,

170.4. LC–MS (ESI) purity 99%, m/z 479.3 [M+H]⁺, (method d). Anal

(C₂₈H₂₈F₆N₆O₇S Â 2.4 H₂O) C, H, N.

IC₅₀values by nonlinear regression according to equation

m = m₀/

(1+[I]/IC₅₀). The K_mvalue for bovine trypsin and Boc-Gln-Ala-Arg-

NH-Mec obtained in triplicate measurements with 9 different

substrate concentrations was 22.12 4.01 lM. The K_mvalue for

human thrombin and Cbz-Gly-Gly-Arg-NH-Mec obtained in dupli-

cate measurements with 12 different substrate concentrations was

40.21 2.33 lM. The K_mvalue for human matriptase-2 in the con-

4.3.71. (R)-3-(N-(1-(4-Amidiniobenzylamino)-1-oxo-3-

phenylpropan-2-yl)sulfamoyl)benzamidinium bis(triﬂuoro-

acetate) (71)

ditioned medium of HEK-MT2 cells and Boc-Gln-Ala-Arg-NH-Mec

obtained in duplicate measurements with nine different substrate

concentrations was 32.18 2.78 lM. K_ivalues were calculated

Yield 22%, mp 235–237 °C. ¹H NMR (DMSO-d₆): d 2.67–2.72 (m,

1H), 2.86–2.90 (m, 1H), 4.01–4.06 (m, 2H), 4.21 (dd, J = 16.1 Hz,

J = 6.0 Hz, 1H), 7.08–7.11 (m, 2H), 7.15–7.17 (m, 3H), 7.19 (d,

J = 8.5 Hz, 2H), 7.66 (dd, J = 7.9 H, J = 7.9 Hz, 1H), 7.69 (d,

J = 8.5 Hz, 2H), 7.83–7.86 (m, 1H), 7.93–7.95 (m, 1H), 7.99 (dd,

J = 1.6 Hz, J = 1.6 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.56 (t,

J = 5.7 Hz, 1H), 9.07, 9.24, 9.31, 9.43 (each s, 8H). APT ¹³C NMR

(DMSO-d₆): d 38.5, 41.8, 58.1, 126.2, 126.6, 126.8, 127.3, 128.1,

128.3, 129.0, 129.4, 130.0, 131.3, 131.9, 136.8, 141.9, 145.4, 158.7

(q, J = 31.0 Hz), 165.0, 165.5, 170.4. LC–MS (ESI) purity 99%, m/z

479.3 [M+H]⁺, (method d). Anal (C₂₈H₂₈F₆N₆O₇S Â 1.2 H₂O) C, H, N.

using the equation K_i= IC₅₀/(1+[S]/K_m).

4.4.2. Bovine trypsin inhibition assay

Assay buffer was 50 mM Tris–HCl, 150 mM NaCl, pH 8.0. The

enzyme stock solution (1 mg/mL) was prepared in 1 mM HCl, kept

at 0 °C and diluted with assay buffer. A 10 mM stock solution of the

ﬂuorogenic substrate Boc-Gln-Ala-Arg-NH-Mec in DMSO was di-

luted with assay buffer. The ﬁnal concentration of the substrate

was 40

mL. Into each well containing 168.8

itor solution in DMSO and 10 L of a substrate solution (800

were added and thoroughly mixed. The reaction was performed

M, of DMSO was 6% and of bovine trypsin was 12.5 ng/

L buffer, 11.2 L of an inhib-

4.3.72. (S)-3-(N-(1-(4-Amidiniobenzylamino)-3-benzyloxy-1-

oxopropan-2-yl)sulfamoyl)benzamidinium bis(triﬂuoro-

acetate) (72)

at 25 °C, initiated by adding 10 lL of enzyme solution (250 ng/

mL) and followed over 400 s.

Yield 7%, mp 207–210 °C. ¹H NMR (DMSO-d₆): d 3.44–3.53 (m,

2H), 4.09 (dt, J = 8.2 Hz, J = 6.3 Hz, 1H), 4.17, 4.30 (each dd,

J = 16.1 Hz, J = 5.7 Hz, 2H), 4.35 (d, J = 7.6 Hz, 2H), 7.22–7.24 (m,

4.4.3. Human thrombin inhibition assay

Assay buffer was 50 mM Tris–HCl, 150 mM NaCl, pH 8.0. The

enzyme stock solution (10000 U/mL) was prepared in water, kept

6504

S. Dosa et al. / Bioorg. Med. Chem. 20 (2012) 6489–6505

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was 40

mL. Into each well containing 173.8

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were added and thoroughly mixed. The reaction was performed

at 25 °C, initiated by adding 5 lL of enzyme solution (100 U/mL)

and followed over 400 s.

M, of DMSO was 6% and of human thrombin was 2.5 U/

L buffer, 11.2 L of an inhib-

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Assay buffer was 50 mM Tris–HCl, 150 mM NaCl, pH 8.0. The

conditioned medium of HEK-MT2 cells was used as a source of

matriptase-2 activity. The conditioned medium was collected and

concentrated, and the supernatant was stored at À20 °C. After

thawing, it was diluted with assay buffer (1:50) and kept at

0 °C.^38,39A 10 mM stock solution of ﬂuorogenic substrate Boc-

Gln-Ala-Arg-NH-Mec in DMSO was diluted with assay buffer. The

ﬁnal concentration of the substrate was 40

6%. Into each well containing 143.8 L buffer, 11.2

itor solution in DMSO and 10 L of a substrate solution (800

were added and thoroughly mixed. At 37 °C the reaction was initi-

M and of DMSO was

L of an inhib-

ated by adding 35

lL of diluted conditioned medium and followed

over 400 s.

21. Schweinitz, A.; Stürzebecher, A.; Stürzebecher, U.; Schuster, O.; Stürzebecher,

J.; Steinmetzer, T. Med. Chem. 2006, 2, 349.

Acknowledgments

22. (a) Stürzebecher, J.; Vieweg, H.; Steinmetzer, T.; Schweinitz, A.; Stubbs, M. T.;

Renatus, M.; Wikström, P. Bioorg. Med. Chem. Lett. 1999, 9, 3147; (b) Verner, E.;

Katz, B. A.; Spencer, J. R.; Allen, D.; Hataye, J.; Hruzewicz, W.; Hui, H. C.;

Kolesnikov, A.; Li, Y.; Luong, C.; Martelli, A.; Radika, K.; Rai, R.; She, M.; Shrader,

W.; Sprengeler, P. A.; Trapp, S.; Wang, J.; Young, W. B.; Mackman, R. L. J. Med.

Chem. 2001, 44, 2753; (c) Schweinitz, A.; Steinmetzer, T.; Banke, I. J.; Arlt, M. J.;

Stürzebecher, A.; Schuster, O.; Geissler, A.; Giersiefen, H.; Zeslawska, E.; Jacob,

U.; Krüger, A.; Stürzebecher, J. J. Biol. Chem. 2004, 279, 33613.

23. (a) Steinmetzer, T.; Schweinitz, A.; Stürzebecher, A.; Dönnecke, D.; Uhland, K.;

Schuster, O.; Steinmetzer, P.; Müller, F.; Friedrich, R.; Than, M. E.; Bode, W.;

Stürzebecher, J. J. Med. Chem. 2006, 49, 4116; (b) Steinmetzer, T.; Dönnecke, D.;

Korsonewski, M.; Neuwirth, C.; Steinmetzer, P.; Schulze, A.; Saupe, S. M.;

Schweinitz, A. Bioorg. Med. Chem. Lett. 2009, 19, 67; (c) Schweinitz, A.;

Dönnecke, D.; Ludwig, A.; Steinmetzer, P.; Schulze, A.; Kotthaus, J.; Wein, S.;

Clement, B.; Steinmetzer, T. Bioorg. Med. Chem. Lett. 2009, 19, 1960; (d)

Hammami, M.; Rühmann, E.; Maurer, E.; Heine, A.; Gütschow, M.; Klebe, G.;

Steinmetzer, T. Med. Chem. Commun. 2012, 3, 807.

This work was supported by the German Research Foundation

(SFB 645). The authors thank Stephanie Hautmann for skillful tech-

nical assistance. V.K. thanks the German Research Foundation (GRK

804). D.H. is supported by a fellowship from the NRW International

Graduate Research School Biotech-Pharma. E.M. thanks Bayer

HealthCare for a fellowship award.

Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.bmc.2012.08.042.

24. Huang, T. L.; Bacchi, C. J.; Kode, N. R.; Zhang, Q.; Wang, G.; Yartlet, N.; Rattendi,

D.; Londono, I.; Mazumder, L.; Vanden Eynde, J. J.; Mayence, A.; Donkor, I. O.

Int. J. Antimicrob. Agents 2007, 30, 555.

25. Huang, T. L.; Vanden Eynde, J. J.; Mayence, A.; Collins, M. S.; Cushion, M. T.;

Rattendi, D.; Londono, I.; Mazumder, L.; Bacchi, C. J.; Yarlett, N. Bioorg. Med.

Chem. Lett 2009, 19, 5884.

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