Novel semicarbazide-derived inhibitors of human dipeptidyl peptidase I (hDPPI)

Bioorganic & Medicinal Chemistry 13 (2005) 4408–4424

Novel semicarbazide-derived inhibitors of human

dipeptidyl peptidase I (hDPPI)

Jon Bondebjerg,^a,*Henrik Fuglsang,^aKirsten Rosendal Valeur,^a

a

Dorte Wissing Kaznelson, Johnny Arnsdorf Hansen, Rene Orup Pedersen,

a

´

Berit Olsen Krogh,^aBo Skaaning Jensen,^aConni Lauritzen,^bGitte Petersen,^b

John Pedersen^band Lars Nærum^a

^aCombio A/S, Vesterbrogade 188, DK-1800 Frederiksberg C, Denmark

^bProzymex A/S, Dr. Neergaards Vej 17, DK-2970 Hørsholm, Denmark

Received 8 February 2005; revised 19 April 2005; accepted 19 April 2005

Available online 12 May 2005

Abstract—Human dipeptidyl peptidase I (hDPPI, cathepsin C, EC 3.4.14.1) is a novel putative drug target for the treatment of

inﬂammatory diseases. Using 1 as a starting point (IC₅₀> 10 lM), we have improved potency by more than 500-fold and success-

fully identiﬁed novel inhibitors of DPPI via screening of a one-bead-two-compounds library of semicarbazide derivatives. Selected

compounds were shown to inhibit intracellular DPPI in RBL-2H3 cells. These compounds were further characterized for adverse

eﬀects on HepG2 cells (cytotoxicity and viability) and their metabolic stability in rat liver microsomes was estimated. One of the

most potent inhibitors, 8 (IC₅₀= 31 3 nM; K_i= 45 2 nM, competitive inhibition), is selective for DPPI over other cysteine

and serine proteases, has a half-life of 24 min in rat liver microsomes, shows approximately 50% inhibition of intracellular DPPI

at 20 lM and is noncytotoxic.

1. Introduction

the solvent. The S2 pocket is deep and hydrophobic,

with a chloride ion and two solvent molecules at the bot-

tom, and Asp 1 at the entrance to anchor the substrate

via an electrostatic interaction. The exclusion domain

blocks extension beyond the S2 pocket, and thus pre-

vents endopeptidase activity. DPPI is synthesized as an

inactive precursor (zymogen), and is activated by a non-

autocatalytic excision of an internal activation peptide

within the N-terminal propeptide.⁷Once activated,

DPPI catalyzes sequential removal of dipeptides from

the N-termini of peptide and protein substrates with

broad speciﬁcity.^3,8,9However, DPPI only cleaves sub-

strates with a free N-terminal amino group, and does

not cleave substrates with P1 or P1⁰Pro, P1 Ile and

P2 ornithine, Lys or Arg.

Human dipeptidyl peptidase I (hDPPI, cathepsin C, EC

3.4.14.1), ﬁrst discovered by Gutmann and Fruton in

1948,¹is a lysosomal cysteine exopeptidase that belongs

to the papain family.^2,3The enzyme is constitutively ex-

pressed in many tissues with highest levels in lung, kid-

ney, liver and spleen. The cloning and sequencing of

cDNAs encoding rat, human and murine DPPI show

that the enzyme is highly conserved.^2,4,5DPPI is a

200 kDa tetramer consisting of four identical subunits,

each composed of three diﬀerent chains: a heavy chain,

a light chain and an exclusion domain.^6a,bThe crystal

structure of DPPI shows that the enzyme has four active

site clefts with the active site residues Cys 234 and His

381 forming an ion pair near the scissile peptide bond.

The S1 pocket is located near the surface, exposed to

DPPI is an important enzyme in lysosomal protein deg-

radation and recent studies in DPPI knock-out mice

show that DPPI functions as a key enzyme in the activa-

tion of granule serine proteases in cytotoxic T lympho-

cytes and natural killer cells (granzymes A and B),^3,10a

mast cells (chymase and tryptase),^10b,cand neutrophils

(cathepsin G, proteinase 3 and elastase).¹¹

Keywords: Human dipeptidyl peptidase I; Inﬂammation; Protease

inhibitors; Semicarbazides.

*

Corresponding author. Tel.: +45 33780018; fax: +45 70222367;

e-mail: job@combio.biz

doi:10.1016/j.bmc.2005.04.048

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

4409

Dominant-negative mutations within the human DPPI

12

whereas azapeptides with reactive leaving groups form

carbamoyl–enzyme complexes that are more stable than

normal acyl–enzymes.²⁶

`

genes are linked to the Papillon-Lefevre syndrome,

a

disease characterized by early periodontitis, palmoplan-

tar hyperkeratosis and a predisposition to bacterial

infections. This indicates that DPPI plays an important

role in the immune system. Recent data¹³show that dis-

ruption of DPPI genes is beneﬁcial for the survival from

sepsis and indicate that DPPI is a potential new target

for the treatment of sepsis. Thus, in combination with

predictive animal models, inhibitors of DPPI will be

beneﬁcial for further studies on the physiological role

of DPPI.

2. Results and discussion

Using 1 (Fig. 1, IC_{50, DPPI}> 10 lM) as a starting point,

we initially screened a 64,000 member unbiased, one-

bead-two-compounds combinatorial library of semicarb-

azides (2). The one-bead-two-compounds library^28–30al-

lows screening of a large number of compounds in a

competitive fashion using biocompatible beads. Each

bead contains an internally quenched ﬂuorogenic sub-

strate and a putative inhibitor. Upon incubation with

a protease the substrate will be cleaved in the presence

of a weak inhibitor, resulting in bright beads under

UV-light due to separation of the quencher from the

immobilized ﬂuorophore. Vice versa, the darkest beads

contain potent inhibitors, which prevent substrate cleav-

age by binding to the protease. Thus, dark beads can be

selected and the identity of the inhibitors (hits) can be

elucidated by MALDI-TOF MS. The hits from the

screening are veriﬁed by resynthesis of the identiﬁed

structure and subsequent IC₅₀determination.

Inhibitors of DPPI include dipeptide diazomethyl

ketones (e.g., Gly-Phe-CH₂N₂, Fig. 1),^14a,ba dipeptide

nitrile,¹⁵dipeptide vinyl sulfones,¹⁶dipeptide acyloxy-

methyl- and ﬂuoromethyl-ketones,¹⁶dipeptide O-acyl

hydroxamic acids,¹⁷arginine-based peptides¹⁸and phos-

phinic tripeptides.¹⁹The naturally occurring cysteine

protease inhibitor E-64 also inhibits DPPI at high

concentrations.²⁰

Herein, we report semicarbazide-derived inhibitors of

DPPI, also known as azapeptides when ﬂanked by ami-

no acid residues.²¹Azapeptides have been reported as

inhibitors of Hepatitis C Virus NS3 protease,^22,23

Human Rhinovirus 3C protease,²⁴papain^25,26and

cathepsins B and K.²⁵Zoladex^ꢁ, an azapeptide ana-

logue of the peptide hormone luliberin (LHRH), is used

for treatment of prostate cancer. Azapeptides contain

one or more amino acids in which the central a-carbon

has been replaced by a nitrogen. Replacement of the P1

a-carbon with a nitrogen decreases the electrophilicity of

the P1 carbonyl group and changes the geometry of the

a-position from tetrahedral to nearly trigonal, resulting

in loss of chirality and an orientation of the side chain

somewhere in between the ^D- and L-conﬁguration of

the corresponding amino acid. Some azapeptides are

metabolically more stable towards enzymatic degrada-

tion than their natural peptide counterparts.²⁷Azapep-

tides form inactivating carbamoyl–enzyme complexes

with cysteine and serine proteases, which undergo slow

hydrolysis.^22,24The rate and extent of acylation–deacyl-

ation depend on the reactivity of the leaving group.²²

Azapeptides with poor leaving groups fail to acylate

the enzyme and hence display competitive inhibition,

Based on the results from the ﬁrst one-bead-two-com-

pounds library screening, 8 (Fig. 1, Table 1, IC₅₀

=

31 3 nM) was synthesized. Subsequently, a second fo-

cused 8000 member library was screened, leading to the

identiﬁcation of 9 (IC₅₀= 21 3 nM), and the design of

10–14. Overall, more than 500-fold increase in potency

was achieved compared to 1.

To reduce both molecular weight and the peptidic char-

acter of the compounds, we substituted the P1⁰–P2⁰

dipeptide moiety with various aromatic groups (Table

2, entries 19–46) leading to the identiﬁcation of 28

(IC₅₀= 560 26 nM) and the more potent analogue 35

(IC₅₀= 123 16 nM).

The selectivity of a range of inhibitors was determined

by screening against a panel of relevant cysteine- and

serine-proteases, as well as CYP450 enzymes. Selected

compounds were also tested in a cellular DPPI assay

using a RBL-2H3 rat mast cell line (Fig. 3), and their

Figure 1. Structures of Gly-Phe-CH₂N₂(or GFCH₂N₂), semicarbazides 1, 8, 28 and the one-bead-two-compounds library 2; F, ﬂuorophore; Q,

quencher.

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J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

Table 1. IC₅₀values for 3–18

Table 1 (continued)

#

R₃

R₂

R₁

IC₅₀(DPPI)

nM

#

R₃

R₂

R₁

IC₅₀(DPPI)

nM

14

445 10

3

622 13

15

16

17

18

219 10

4

5

933 251

108 23

440 64

496 70

378 50

6

7

968 185

10,967 3519

cytotoxicity and viability in HepG2 cells was deter-

mined. The metabolic stability of these compounds

was evaluated in rat liver microsomes.

8

31

21

45

3

4

2.1. Chemistry

For solution-phase synthesis (Scheme 2, 19–44) the

semicarbazide bond was formed by acylation of the

amine with the appropriate 1-acyl-1⁰-carbonylimid-

azolylhydrazide, formed in situ by reaction of the amino

acid hydrazide with CDI. The hydrazides were formed

by in situ activation of the appropriate amino acid with

CDI, followed by reaction with hydrazine. The crude

products were usually used directly in the next step.

When using the solid phase approach (Scheme 1, 3–18)

Fmoc–NHNH₂was used to introduce the semicarbazide

bond via in situ activation with CDI, similar to what

was previously reported.²⁵The Fmoc group could sub-

sequently be removed by standard treatment with piperi-

dine in DMF, followed by acylation at the 1-position

with an activated carboxylic acid derivative (e.g., TBTU

activation). Substituents in the 2-position of the semi-

carbazide functionality can be introduced via substi-

tuted hydrazines (Scheme 3), as described previously.³¹

Thus, benzylhydrazine was selectively Boc-protected

on the 1-position with excess Boc₂O at ꢀ78 ꢂC, followed

by Fmoc-protection of the 2-position with Fmoc–Cl and

DIPEA. Treatment with TFA gave 1-Fmoc-2-benzyl-

hydrazine I in 46% yield after work-up. Reaction with

phosgene in toluene aﬀorded crude 2-(chlorocarbonyl)-

1-Fmoc-2-benzylhydrazine, which was reacted directly

with 2-chlorobenzylamine to give the 1-Fmoc-2-benzyl-

9

10

11

12

39

22

3

2

13

19

6

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

4411

Table 2. IC₅₀values for 19-46

Table 2 (continued)

#

R₄R₅R₆R₇R₈

IC₅₀(DPPI) nM

34 Et

H

600 37

#

R₄

R₅R₆R₇

R₈

IC₅₀(DPPI) nM

8066 252

19 Me

20 Me

H

35 Et

H

123 16

1396 13

13,167 1018

6292 419

21 Me

22 Me

H

36 Et

157 23

37 Et

38 Et

39 Et

H

678 30

530 56

494 29

23 Me

24 Me

H

NI^a

1622 101

25 Et

26 Et

H

2045 277

759 117

40 Et

41 Et

42 Et

43 Bu

44 Bn

45 Et

46

H

Bn

H

986 36

1284 175

684 55

2312 303

1227 167

NI^a

27 Et

28 Et

29 Et

H

Me

H

NI^a

560 26

562 91

H

30 Et

H

5422 117

269 26

31 Et

NI^a

32 Et

H

698 53

^aNo inhibition at 10 lM.

4-(o-chlorobenzyl)semicarbazide II. The Fmoc group

was removed with piperidine and the ﬁnal (2S)-2-amino-

butanoyl substituent was introduced via the DIC-medi-

ated anhydride of (2S)-N-Boc-aminobutyric acid to give

45 after deprotection and puriﬁcation.

33 Et

422 25

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J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

Scheme 1. Solid-phase synthesis of semicarbazides 3–18. Reagents and conditions: (a) Fmoc–AA–OH, TBTU, NEM, DMF; or Fmoc–AA–OPfp,

DhbtOH, DMF; (b) 20% piperidine in DMF; (c) Fmoc–NHNH₂, CDI, DMF; (d) TFA–TIPS 98:2.

methionine was converted to the sulfonium salt III by

condensation with tert-butylcarbazate via activation

with CDI, followed by reaction with methyl iodide.

Cyclization was achieved using NaH, aﬀording IV in

50% yield. Removal of the Boc group with HCl in diox-

ane gave V as the HCl salt, which was converted to the

protected semicarbazide VI by activation with CDI fol-

lowed by displacement with o-chlorobenzylamine. The

Cbz group was removed with HBr in acetic acid to give

Scheme 2. Synthesis of semicarbazides 19–44 in solution. Reagents

and conditions: (a) CDI, THF; (b) NH₂NH₂, THF; (c) R₇R₈NH,

THF; (d) TFA–H₂O 95:5.

46.

All target compounds were puriﬁed by HPLC to a min-

imum purity of 95% as determined by HPLC and char-

1

acterized by H NMR and ES-MS.

2.2. Structure–activity relationships

Starting with the weak inhibitor 1 we initially synthe-

sized and screened a 64,000 member one-bead-two-com-

pounds combinatorial library of semicarbazides, four

amino acids in length in order to explore the P2⁰binding

site as well. The crystal structure of human DPPI⁶re-

vealed that the S2⁰pocket is located in the Gln 228-

Cys 234 loop, however, very little is known about the

Scheme 3. Synthesis of 45. Reagents and conditions: (a) (i) Boc₂O,

DIPEA, CHCl₃, ꢀ78 ꢂC; (ii) Fmoc–Cl, DIPEA; (iii) TFA–H₂O 95:5

P2⁰SAR. Based on the results from the ﬁrst round of li-

brary screening, we synthesized 3–7, and from 4

(IC₅₀= 933 251 nM) it became evident that addition

(46%, three steps); (b) COCl₂, toluene; (c) 2-chlorobenzylamine,

DIPEA, CH₃CN; (d) piperidine; (e) Boc-Abu-OH, DIC, THF; (f)

TFA–H₂O 95:5 (9%, ﬁve steps).

of P2⁰Trp increased the potency >10-fold compared

to 1, indicating that this position can indeed have a sig-

niﬁcant impact on binding. Further analysis revealed

The conformationally constrained analogue 46 was pre-

pared via the pyrrolidinone V (Scheme 4) following a

previous procedure,³²although experimental details

and analytical data were not reported. Thus, Cbz-^L-

that for the P2 residue the preference is

ethyl > methyl ꢁ piperidinyl (4, 5 and 7), consistent

with previous ﬁndings that dipeptides with small

aliphatic P2 groups are optimal substrates, and a free

Scheme 4. Synthesis of 46. Reagents and conditions: (a) CDI, THF; then BocNHNH₂(96%); (b) MeI (100%); (c) NaH, DMF, DCM (50%); (d) HCl

in dioxane (49%); (e) CDI, THF, o-chlorobenzylamine (34%); (f) HBr in acetic acid (83%).

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

4413

(primary) terminal amino group is crucial.⁸The latter

was also conﬁrmed by various mono- and di-N-alkyla-

tions of 1, which resulted in complete loss of activity

at 20 lM (S1–S6, Table S1; see Supplementary data).

The former was conﬁrmed by comparing 20

(IC₅₀= 1396 13 nM), 26 (IC₅₀= 759 117 nM), 43

(IC₅₀= 2312 303 nM) and 44 (IC₅₀= 1227 167 nM),

although the benzyl- and methyl-analogues (20 and 44)

were surprisingly equally potent. However, the p-bro-

mobenzyl analogue S42 (Table S2) was only weakly ac-

tive at 10 lM, indicating that the bottom of the S2

pocket might have been reached. P2 isopropyl (S39)

and 3-thiabutyl (S41) were also tolerated. The conform-

ationally constrained analogue 46 was inactive at

10 lM, as was the R-isomer (data not shown).

being the best in the series. In an attempt to rapidly fur-

ther explore the SAR in the ortho position, we prepared

a series of anilides (S44–S57, Table S3). However, judg-

ing from the resulting 15- to 1000-fold loss in potency

compared to 35, this was not a well tolerated modiﬁca-

tion. In contrast, the thioether 36 (IC₅₀= 157 23 nM)

retained its potency, indicating that rather bulky and

ﬂexible groups are tolerated, although it appears that

planarity is potentially important (33; IC₅₀

=

422 25 nM and 34; IC₅₀= 600 37 nM). Although

35, the most potent compound in this series, is at least

5-fold less potent than the best compounds from the

ﬁrst series (3–18), further optimization should be

feasible.

2.3. Reversibility, selectivity, metabolic stability and

cellular activity

Intrigued by the fact that 3 (IC₅₀= 622 13 nM) with

P2 cyclohexyl and 6 (IC₅₀= 968 185 nM) with P2 pyr-

rolidine, both with P1⁰homophenylalanine, were sur-

The reversibility of inhibition of DPPI by 8 was investi-

gated by comparing its kinetics with the irreversible

inhibitor Gly-Phe-CH₂N₂(GFCH₂N₂, Fig. 2).³³During

the ﬁrst 600 s of the reaction with an initial substrate

concentration of 150 lM (1 · K_m), the velocity of the

substrate cleavage in the presence of 150 nM 8 was mea-

sured as lower (2.4 RFU/s) than the measured velocity

of the substrate cleavage in the control reaction

(6.6 RFU/s). After addition of substrate to 1.5 mM

(10 · K_m, at 6000 s), the velocity of substrate cleavage

in the presence of 150 nM 8 was equal to the velocity

of substrate cleavage in the control reaction (approxi-

mately 11 RFU/s for both reactions, in the interval

6000–7000 s), indicating reversible inhibition. At the

same high substrate concentration, 20 lM of the inhibi-

tor GFCH₂N₂reduced the velocity of substrate cleav-

age signiﬁcantly (a reduction from 11 to 2 RFU/s),

indicating an irreversible mode of inhibition for this

prisingly

potent

considering

their

seemingly

unfavourable P2 group, we synthesized 8 giving a signiﬁ-

cant boost in potency. We then aimed at increasing po-

tency by screening a second, 8000 member library

focussed around the general P2–P1⁰–P2⁰motif ꢀsmall ali-

phatic–aromatic–aromaticꢁ. This resulted in the identiﬁ-

cation of 9 with a minor increase in potency. Attempts

to optimize the P2⁰substituent further did not lead to

a signiﬁcant increase in potency (10–14). With respect

to the P1⁰residue hydrophobic, aromatic groups were

generally well tolerated, consistent with the shallow

and hydrophobic nature of the S1⁰pocket,⁶although

homophenylalanine was far superior to both p-chloro-

phenylalanine, Trp, 2-naphthylalanine and p-phenyl-

phenylalanine, when comparing the screening hits 14

(IC₅₀= 445 10 nM), 16 (IC₅₀= 108 23 nM), 17

(IC₅₀= 440 64 nM) and 18 (IC₅₀= 496 70 nM) to 8

(IC₅₀= 31 3 nM) and 11 (IC₅₀= 39 3 nM). Discour-

aged by the complete lack of activity at 10 lM for 45

with P1 benzyl, a preferred substrate P1 side chain,⁸

no attempts were made towards introducing further P1

diversity. It might be speculated that P1 substitution

of the semicarbazides results in an unfavourable orienta-

tion of the side chain due to the resulting nearly trigonal

geometry. It is known that ^D-amino acids are not toler-

ated at this position.⁸However, an electronic eﬀect can-

not be ruled out since N-substitution decreases the

electrophilicity of the semicarbazide carbonyl, thus

aﬀecting the putative interaction with the active site

cysteine.

In an attempt to reduce molecular weight and the num-

ber of rotatable bonds, a series of less peptidic semicarb-

azides were prepared (19–44). In this series P1⁰benzyl

(19;

2045 277 nM) and 1-naphthylmethyl (24; IC₅₀

IC₅₀= 8066 252 nM

and

25;

IC₅₀

=

1622 101 nM and 42; IC₅₀= 684 55 nM) was ini-

tially identiﬁed as suitable groups. Benzyl was selected

as a starting point for potency optimization due to the

wide variety of commercially available benzylamines,

thus facilitating rapid parallel synthesis. The preferred

aromatic monosubstitution o > p > m (20–22) was

quickly identiﬁed. In the ortho position, many groups

were tolerated, with phenyl (35; IC₅₀= 123 16 nM)

Figure 2. Reversibility studies using 150 nM 8 and 20 lMGFCH₂N₂at

150 lM substrate concentration (left-hand side). Addition of saturat-

ing substrate (1.5 mM, 10 · K_m; right-hand side) restores enzymatic

activity in the presence of 8, indicating reversible inhibition, whereas

GFCH₂N₂inhibits the enzyme in an irreversible manner. The

experiment was performed twice.

4414

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

inhibitor.³⁴The lack of inhibition with GFCH₂N₂in the

initial period (600 s) is presumably because this inhibitor

requires a certain time period to irreversibly occupy all

the substrate binding sites in the enzyme. In the period

from 4000 to 6000 s, GFCH₂N₂completely inhibits sub-

strate cleavage despite the fact that there is still nonc-

leaved substrate present in the solution (i.e.,

comparing the absolute ﬂuorescence value of approxi-

mately 18,000 RFU to the maximal ﬂuorescence value

of approximately 23,000 RFU reached in the control

reaction), further indicating an irreversible mode of inhi-

bition for GFCH₂N₂. The sudden decrease in ﬂuores-

cence signal at 6000 s is caused by substrate quenching

due to the high amount of substrate added to the reac-

tions (10 · K_m). This quenching phenomenon is known

as the inner ﬁlter eﬀect.³⁵

tion of the cysteine proteases cathepsin B and H. No

inhibition of the cysteine protease cathepsin L was ob-

served. Compounds 28 and 42–44 all displayed

IC₅₀> 10 lM against cathepsins B, H and L. Selected

compounds were also screened against a panel of cyto-

chrome P450 enzymes (Table 4). Only the peptidic series

of semicarbazides 8–13 and 16 showed signiﬁcant inhibi-

tion. For all seven compounds this eﬀect was observed

with respect to CYP3A4, the major cytochrome respon-

sible for metabolism of nearly 50% of marketed drugs.³⁷

Whether CYP3A4 was inhibited by the tested com-

pounds or whether these compounds were metabolized

by CYP3A4, thereby causing apparent inhibition, was

not investigated further. The metabolic stability of 8,

9, 16, 28, 38 and 39 was determined in rat liver micro-

somes. All compounds except 39 were signiﬁcantly

metabolized after 15 min with half-lives in the range of

17–24 min (Table 5). Finally, the eﬀect of 3, 8, 26, 28

and 42–44 was tested in a cellular DPPI assay using a

RBL-2H3 rat mast cell line (Fig. 3). Several compounds

displayed cellular eﬃcacy toward DPPI at 20 lM with 8

and 42 being the most potent in the series, although both

were less potent than the irreversible inhibitor

GFCH₂N₂. In addition, 3, 8, 26, 28 and 42–44 showed

neither cytotoxicity (HepG2 cells; LDH release ꢂ50%

Selected compounds from each series were screened for

their eﬀect on other proteases (Table 3). No inhibition

of the serine proteases cathepsin G, neutrophil elastase,

tryptase and chymase were observed at 10 lM for any of

the compounds. In addition, compounds 3, 8, 26, 28, 39

and 42–44 did not inhibit the serine protease dipeptidyl

peptidase IV³⁶(DPPIV) at 10 lM (data not shown).

However, 8–13 all displayed moderate to weak inhibi-

Table 3. Selectivity screening^a

#

Cat B

Cat G

Cat H

3200 581

2182 383

Cat L

N. elastase

Tryptase

Chymase

8

4500 275

927 47

>10,000

ND

9

10

11

12

13

28

42

43

44

6499 997

6992 832

2602 186

2200 272

>10,000

ꢃ10,000^b

3152 428

7341 897

1000–10,000^c

>10,000

ND

^aIC₅₀values or estimated IC₅₀values (from cut-oﬀ determinations) in nM.

^bCut-oﬀ at 10 lM showed approximately 50% inhibition (double determination).

^cBased on cut-oﬀ at 1 lM and 10 lM (double determinations); ND—not determined.

Table 4. Cytochrome P450 inhibition^a

#

CYP1A2

CYP2C9

CYP2C19

CYP2D6

CYP3A4

3

>20,000

>10,000

>5000

>20,000

>10,000

>20,000

>10,000

>5000

>20,000

2274 174

>20,000

>1500

8

593 485

984 121

100–500^b

898 236

>20,000

9

10

11

12

13

16

26

28

35

42

43

44

17,335 8886

4749 3700

>10,000

>20,000

>15,000

>1500

^aIC₅₀values or estimated IC₅₀values (from cut-oﬀ determinations) in nM.

^bCut-oﬀ at 100 and 500 nM concentrations, determined in doublets.

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

4415

Table 5. Metabolic stability^a

#

8

9

16

28

38

39

b

%

69(24)

65(20)

58(17)

59(22)

55

85

^aIn rat liver microsomes. All values are the mean of two determinations.

^b% remaining after 15 min; t_1/2in minutes given in parentheses. The t_1/2values for reference compounds imipramine, verapamil and terfenadine are 5,

24 and 20 min, respectively.

which may serve to further validate DPPI as a potential

drug target in inﬂammation.

4. Experimental

4.1. Abbreviations

AA, amino acid; Abz, 2-aminobenzoyl; AFC, 7-amino-

triﬂuoromethyl coumarin; AMC, 7-amido-4-methyl-

coumarin; Boc, tert-butyloxycarbonyl; CDI, 1,1⁰-car-

Figure 3. Inhibition of DPP-I in total cell lysate from a rat mast cell

line (RBL-2H3) incubated in the presence of 20 lM 3, 8, 26, 28 or 42–

44 for 30 min. DMSO was used as control.

bonyldiimidazole; DCM, dichloromethane; DE,

diethylether; DhbtOH, 3,4-dihydro-3-hydroxy-4-oxo-

1,2,3-benzotriazine; DIPEA, diisopropylethylamine;

DMF, N,N-dimethylformamide; DTNB, 5,5⁰-dithio-

bis(2-nitrobenzoic acid); DTT, 1,4-dithiothreitol;

at 100 lM) nor reduced viability (HepG2 cells; viable

cells ꢁ50% at 100 lM).

EtOAc, ethyl acetate; EDTA, ethylenediaminetetraace-

tic acid; Fmoc, 9-ﬂuorenylmethyloxycarbonyl; MAL-

DI-TOF MS, matrix assisted laser desorption

ionization time-of-ﬂight mass spectrometry; Im, 1-imid-

azolyl; NEM, N-ethylmorpholine; PE, petroleums ether

(bp 60–80 ꢂC); Pfp, pentaﬂuorophenyl; PEGA, poly(eth-

ylene glycol)–poly(acryl amide) copolymer; Rink amide

linker, p-[(R,S)-a-[1-(9H-ﬂuoren-9-yl)-methoxyformam-

ido]-2,4-dimethoxybenzyl]-phenoxyacetic acid; TBTU,

N-[(1H-benzotriazol-1-yl)-(dimethylamino)-methylene]-

N-methylmethan-aminium tetraﬂuoroborate N-oxide;

TFA, triﬂuoroacetic acid; THF, tetrahydrofuran; TIPS,

triisopropylsilane; TMSBr, trimethylsilyl bromide; One-

and three-letter codes are used for the amino acids

according to IUPAC.

3. Conclusion

Starting with the weak semicarbazide-based inhibitor 1,

we have identiﬁed novel DPPI inhibitors based on a

semicarbazide scaﬀold, using a one-bead-two-com-

pounds library approach to aid our design. In P2, ami-

nobutyric acid was preferred, and for the peptidic

semicarbazides 3–18 homophenylalanine was preferred

in P1⁰, whereas the best of the nonpeptidic analogues

19–46 had substituted benzylic groups in this position

with the ortho position being favoured. In P2⁰several

aromatic groups were tolerated. Introduction of a ben-

zyl group in P1 gave rise to loss of activity, as did the

introduction of conformational constraint in P2. In a

selectivity study with other cysteine and serine proteases,

the inhibitors were selective for DPPI, although moder-

ate to weak inhibition of cathepsins B and H was ob-

served for the peptidic semicarbazides. The same

compounds also showed signiﬁcant inhibition of

CYP3A4. The half-lives in rat liver microsomes were

around 20 min for selected compounds and several

inhibitors showed cellular eﬃcacy towards DPPI. None

of these compounds was cytotoxic or inﬂuenced cell via-

bility. One of the best inhibitors was 8, with an

IC₅₀= 31 3 nM and K_i= 45 2 nM against DPPI.

The compound showed competitive, reversible inhibi-

tion. Although the nonpeptidic semicarbazides were

generally less potent, the discovery of 35

(IC₅₀= 123 16 nM) indicates that further optimiza-

tion should be feasible. X-ray crystallography studies

with these inhibitors are on-going and the results will

be reported elsewhere. The results reported herein can

be used to further optimize inhibitors of DPPI with re-

spect to potency, selectivity and pharmacokinetics,

4.2. Materials and methods

Experiments were conducted at room temperature

(20 ꢂC), unless otherwise noted. All solvents were HPLC

grade. Anhydrous solvents were obtained by storing

˚

over 4 A activated molecular sieves. Unless otherwise

noted, starting materials were purchased from commer-

cial suppliers and used without further puriﬁcation.

Yields were generally of puriﬁed product and were not

optimized.

Solid phase reactions run at room temperature were per-

formed in ﬂat-bottom polyethylene syringes equipped

with sintered Teﬂon ﬁlters (70 lm pores), Teﬂon tubing,

Teﬂon valves for ﬂow control and suction to drain the

syringes from below, or on a VacMaster parallel synthe-

sis rack. Fmoc deprotection was performed with 20%

(v/v) piperidine in DMF (2 + 10 min). TBTU couplings

were performed by dissolving the acid (3 equiv) in

DMF with NEM (4 equiv), followed by addition of

TBTU (2.88 equiv). The resulting solution was preacti-

vated for 10 min before use. The reactions were

4416

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

monitored using the Kaiser test.³⁸Pfp esters (3 equiv)

were coupled with DhbtOH (1 equiv) present. The dis-

appearance of the bright yellow colour indicated com-

plete capping of the resin-bound amino groups. Solid

phase reactions were generally run in an amount of sol-

vent that was suﬃcient to cover the resin. Resin loadings

were determined by spectrophotometric analysis at

290 nm of the dibenzofulvene–piperidine adduct formed

upon Fmoc deprotection, using a Perkin–Elmer Lamb-

da 7 UV/vis spectrometer and a calibration curve from

a known sample.

4.3.1. 1-(1-Aminocyclohexane-1-carbonyl)-4-{2S-N-[2S-

3-(m-ﬂuorophenyl)propan-2-yl-amide]-4-phenylbutan-2-yl-

amide}semicarbazide (3). Fmoc–Rink–PEGA₈₀₀resin

(1.6 g, L = 0.25 mmol/g, 0.4 mmol) was Fmoc deprotec-

ted and washed with DMF (·5). Fmoc-3-ﬂuorophenyl-

alanine (487 mg, 1.2 mmol) was coupled using TBTU

(370 mg, 1.15 mmol) and NEM (203 lL, 1.59 mmol) in

dry DMF (12 mL). The resin was washed with DMF

(·5), Fmoc deprotected and washed with DMF (·5).

Fmoc–homophenylalanine (482 mg, 1.2 mmol) was like-

wise coupled using TBTU and NEM. Following a cycle

of wash (DMF · 5), deprotection and wash (DMF · 5)

the semicarbazide bond was formed by overnight reac-

tion with Fmoc–NHNH–COIm, formed from Fmoc–

NHNH₂(302 mg, 1.2 mmol) and CDI (193 mg,

1.2 mmol) in dry DMF (3 mL, 2 h preactivation, then

the resulting solution was added to the resin along with

further 9 mL dry DMF). At this point a Kaiser test was

found slightly positive and the coupling was repeated.

The resin was washed with DMF (·5) and DCM (·3)

and dried in vacuo. A portion of the resin (1294 mg,

318 lmol) was subjected to another cycle of wash

(DMF · 5), deprotection and wash (DMF · 5), and

then Boc-1-amino-1-cyclohexane carboxylic acid

(232 mg, 954 lmol) was coupled using TBTU (294 mg,

916 lmol) and NEM (161 lL, 1.27 mmol) in DMF

(10 mL). The resin was washed with DMF (·5) and

DCM (·3) and treated with neat TFA–TIPS 98:2 for

1 h and twice for 30 min. The combined fractions were

concentrated and the residue was puriﬁed by HPLC to

give the title compound as a white solid. Yield: 100 mg

(60%); HPLC (1): t_R= 4.21 min (>99%); HPLC (2):

t_R= 7.00 min (>98%);¹H NMR (DMSO-d₆/D₂O 10:1,

250 MHz): d 7.33–6.95 (m, 9H), 4.50–4.44 (m, 1H),

4.13–4.07 (m, 1H), 3.09–3.02 (dd, J = 5.0, 13.8, 1H),

2.88–2.79 (dd, J = 9.7, 13.8, 1H), 2.53–2.44 (2H, par-

tially under DMSO signal), 2.07–1.99 (m, 2H), 1.87–

1.14 (m, 10H); ES-MS: mass calcd for C₂₇H₃₆FN₆O₄

527.3 (MH⁺). Found m/z 527.2.

NMR spectra were acquired on a Bruker Advance DRX

250. Chemical shifts are reported in ppm, relative to

internal solvent peaks (2.49 for DMSO-d₆, 7.25 for

CDCl₃, 4.75 for D₂O, 3.35 for CD₃OD). Coupling con-

stants J are reported in hertz. All key compounds were

characterized by NMR and for several of the target

semicarbazides the presence of rotamers was evident

from isomeric resonances.

ES-MS spectra were obtained on a Micromass Quattro

micro^TMinstrument in the positive mode. Analytical

HPLC was performed on a Gilson system (UV/vis-155

detector at 215 and 254 nm, 402 syringe pump, 819

injection module, valvemate 35, 864 degasser, 233 XL

on-line column switching module, and a Zorbax 300SB

RP-18 column, 4.6 · 50 mm with a 322 pump), using

two eluent systems. System 1: Eluents A (0.1% TFA in

water) and B (0.1% TFA in acetonitrile) were used in

a linear gradient (0% B ! 100% B in 7 min) with a ﬂow

of 1 mL/min. System 2: Eluents A (0.1% TFA in water)

and B (0.1% TFA in acetonitrile) were used in a linear

gradient (0% B ! 90% B in 10 min) with a ﬂow of

0.5 mL/min. Purity (given in parentheses) is at 215 nm.

Preparative HPLC was performed on the same Gilson

system, using a Zorbax 300SB RP-18, 21.2 mm · 25 cm

column, with a ﬂow of 15 mL/min.

TLC plates used were Merck silica gel 60F₂₅₄on alumin-

ium. Visualization was achieved with UV light when

applicable, or developed by staining with the AMC re-

agent (21 g (NH₄)₆Mo₇O₂₄, 1 g Ce(SO₄)₂, 31 mL

H₂SO₄, 500 mL water) or phosphormolybdic acid

(2.5 g phosphormolybdic acid in 500 mL ethanol).

4.3.2. 1-(2S-2-Aminopropanoyl)-4-{2S-N-[2S-3-(indol-3-

yl)propan-2-yl-amide]-3-phenylpropan-2-yl-amide}semi-

carbazide (4). Following the procedure for 3 with the

appropriate starting materials. White solid. Yield:

13.1 mg (44%); HPLC (1): t_R= 3.56 min (>99%); HPLC

(2): t_R= 6.36 min (>98%); ¹H NMR (D₂O, 250 MHz): d

(rotamers observed) 7.69–7.66 (d, J = 7.6, 1H), 7.42–

7.39 (d, J = 7.9, 1H), 7.31–7.02 (m, 8H), 4.56–4.41 (m,

2H), 3.91–3.75 (1H, partially under water signal),

3.24–3.16 (dd, J = 5.3, 14.6, 1H), 3.09–2.99 (m, 2H),

2.86–2.77 (dd, J = 8.1, 13.8, 1H), 1.44–1.41 (d, J = 7.0,

3H); ES-MS: mass calcd for C₂₄H₃₀N₇O₄480.2

(MH⁺). Found m/z 480.1.

Elemental analysis was performed by Microanalytisches

Laboratorium, University of Vienna (Austria). Evalua-

tion of compound metabolic stability in rat liver micro-

somes was performed by Cerep (Seattle).

4.3. Synthesis of inhibitors 3–18

Compounds 3–18 were all prepared using TentaGel S-

NH₂(L = 0.41 mmol/g, 150 mg, 0.062 mmol) or

PEGA₈₀₀resin derivatized with the Rink amide linker.

Coupling of individual amino acid derivatives were per-

formed using TBTU or Pfp-ester chemistry, as described

in Section 4.2. The semicarbazide bond was formed

using Fmoc–NHNH–COIm, as described in 3. Com-

pounds 9–18 were prepared by Schafer-N (Copenhagen,

Denmark).

4.3.3. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(indol-3-yl)-

propan-2-yl-amide]-3-phenylpropan-2-yl-amide}semicarb-

azide (5). Following the procedure for 3 with the appro-

priate starting materials. White solid. Yield: 13.3 mg

(44%); HPLC (1): t_R= 3.64 min (>99%); HPLC (2):

t_R= 6.41 min (>98%); ¹H NMR (D₂O, 250 MHz): d

7.66–7.63 (d, J = 7.6, 1H), 7.52–7.49 (d, J = 8.0, 1H),

7.34–7.13 (m, 6H), 7.01–6.97 (m, 2H), 4.61–4.56 (m,

1H), 4.35–4.29 (t, J = 7.4, 1H), 4.02–3.96 (t, J = 6.6,

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

4417

1H), 3.36–3.28 (dd, J = 5.9, 14.7, 1H), 3.19–3.10 (dd,

J = 8.4, 14.7, 1H), 2.88–2.77 (m, 2H), 1.99–1.87 (m,

2H), 1.04–0.98 (t, J = 7.6, 3H); ES-MS: mass calcd for

C₂₅H₃₂N₇O₄494.2 (MH⁺). Found m/z 494.

8 mg (15%); HPLC (1): t_R= 4.35 min (>99%); HPLC

(2): t_R= 7.37 min (>99%); ¹H NMR (CD₃OD,

250 MHz): d 7.47–7.24 (m, 9H), 4.86–4.79 (dd, J = 5.3,

9.5, 1H), 4.35–4.27 (dd, J = 5.4, 8.1, 1H), 4.04–3.96

(app t, J = 6.5, 1H), 3.45–3.36 (dd, J = 5.3, 14.0, 1H),

3.15–3.06 (dd, J = 9.6, 14.0, 1H), 2.78–2.66 (t, J = 8.4,

2H), 2.19–1.95 (m, 4H), 1.30–1.20 (t, J = 7.5, 3H); ES-

MS: mass calcd for C₂₄H₃₂ClN₆O₄503.2 (MH⁺). Found

m/z 503.4.

4.3.4. 1-(2S-Pyrrolidin-2-carbonyl)-4-{2S-N-[2S-3-(indol-

3-yl)propan-2-yl-amide]-4-phenylbutan-2-yl-amide}semi-

carbazide (6). Following the procedure for 3 with the

appropriate starting materials. White solid. Yield:

16.2 mg (51%); HPLC (1): t_R= 3.90 min (>99%); HPLC

(2): t_R= 6.79 min (>97%); ¹H NMR (DMSO-d₆/D₂O

10:1, 250 MHz): d 7.72–7.69 (d, J = 7.6, 1H), 7.44–7.04

(m, 9H), 4.62–4.57 (m, 1H), 4.32–4.21 (m, 2H), 3.38–

3.03 (m, 4H), 2.63–2.57 (1H, partially under DMSO sig-

nal), 2.43–2.36 (m, 1H), 2.08–1.80 (m, 6H); ES-MS:

mass calcd for C₂₇H₃₄N₇O₄520.3 (MH⁺). Found m/z

520.1.

4.3.9. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(m-meth-

oxyphenyl)propan-2-yl-amide]-4-phenylbutan-2-yl-amide}-

semicarbazide (11). Following the procedure for 3 with

the appropriate starting materials. White solid. Yield:

23 mg (46%); HPLC (1): t_R= 4.03 min (>99%); HPLC

(2): t_R= 6.87 min (>99%); ¹H NMR (CD₃OD,

250 MHz): d 7.51–7.30 (m, 6H), 7.04–7.02 (m, 2H),

6.92–6.88 (m, 1H), 4.88–4.82 (dd, J = 5.1, 9.7, 1H),

4.32–4.27 (dd, J = 5.3, 7.9, 1H), 4.05–4.00 (app t,

J = 6.5, 1H), 3.89 (s, 3H), 3.15–3.05 (dd, J = 5.1, 13.9,

1H), 2.77–2.63 (dd, J = 9.7, 13.9, 1H), 2.20–1.95 (m,

4H), 1.31–1.20 (t, J = 7.5, 3H); ES-MS: mass calcd for

C₂₅H₃₅N₆O₅499.3 (MH⁺). Found m/z 499.4.

4.3.5. 1-(2S-Piperidin-2-carbonyl)-4-{2S-N-[2S-3-(indol-3-

yl)propan-2-yl-amide]-3-phenylpropan-2-yl-amide}semi-

carbazide (7). Following the procedure for 3 with the

appropriate starting materials. White solid. Yield:

13.7 mg (43%); HPLC (1): t_R= 3.58 min (>99%); HPLC

(2): t_R= 6.47 min (>97%); ¹H NMR (DMSO-d₆/D₂O

10:1, 250 MHz): d 8.16–8.12 (d, J = 8.1, 1H), 7.61–7.58

(d, J = 7.6, 1H), 7.34–7.31 (d, J = 7.9, 1H), 7.23–6.96

(m, 7H), 4.51–4.34 (m, 2H), 3.75–3.59 (1H, partially un-

der water signal), 3.23–2.70 (m, 6H), 2.08–1.21 (m, 6H);

ES-MS: mass calcd for C₂₇H₃₄N₇O₄520.3 (MH⁺).

Found m/z 520.1.

4.3.10. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(benzo[b]-

thiophen-3-yl)propan-2-yl-amide]-4-phenylbutan-2-yl-amide}-

semicarbazide (12). Following the procedure for 3 with

the appropriate starting materials. White solid. Yield:

8 mg (16%); HPLC (1): t_R= 4.48 min (>99%); HPLC

(2): t_R= 7.52 min (>99%); ¹H NMR (CD₃OD,

250 MHz): d 7.99–7.92 (d, J = 7.9, 1H), 7.86–7.83 (d,

J = 7.6, 1H), 7.45–7.11 (m, 8H), 4.91–4.72 (m, partially

under water signal, 1H), 4.15–4.09 (dd, J = 5.3, 8.1,

1H), 3.88–3.83 (app t, J = 6.5, 1H), 3.62–3.47 (dd,

J = 5.3, 14.8, 1H), 3.35–3.19 (m, partially under metha-

nol signal, 1H), 2.56–2.46 (t, J = 8.3, 2H), 2.06–1.71 (m,

4H), 1.14–1.03 (t, J = 7.5, 3H); ES-MS: mass calcd for

C₂₆H₃₃N₆O₄S 525.2 (MH⁺). Found m/z 525.4.

4.3.6. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(m-ﬂuoro-

phenyl)propan-2-yl-amide]-4-phenylbutan-2-yl-amide}semi-

carbazide (8). Following the procedure for 3 with the

appropriate starting materials. White solid. Yield:

21.6 mg (54%); HPLC (1): t_R= 4.17 min (>99%); HPLC

(2): t_R= 6.95 min (>97%); ¹H NMR (D₂O, 250 MHz): d

7.22–6.72 (m, 9H), 4.53–4.45 (m, 1H), 3.92–3.81 (m,

2H), 3.15–3.07 (dd, J = 5.3, 14.4, 1H), 2.85–2.75 (dd,

J = 10.4, 14.4, 1H), 2.32–2.26 (t, J = 7.8, 2H), 1.88–

1.59 (m, 4H), 0.92–0.81 (t, J = 7.5, 3H); ES-MS: mass

calcd for C₂₄H₃₂FN₆O₄487.24 (MH⁺). Found m/z

487.2. Anal. (C₂₄H₃₁FN₆O₄Æ1H₂OÆ1CF₃COOH) C, H,

N: Calcd 50.48, 5.54, 13.59; found, 50.59, 5.36, 13.42.

4.3.11. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(2-naph-

thyl)propan-2-yl-amide]-4-phenylbutan-2-yl-amide}semi-

carbazide (13). Following the procedure for 3 with the

appropriate starting materials. White solid. Yield:

5 mg (10%); HPLC (1): t_R= 4.50 min (>98%); HPLC

(2): t_R= 7.61 min (>99%); ¹H NMR (CD₃OD,

250 MHz): d 7.85–7.74 (m, 4H), 7.46–7.37 (m, 3H),

7.23–6.90 (m, 5H), 4.91–4.77 (m, partially under water

signal, 1H), 4.12–4.07 (dd, J = 5.6, 7.9, 1H), 3.87–3.82

(app t, J = 6.6, 1H), 3.49–3.41 (dd, J = 5.1, 13.8, 1H),

3.19–3.09 (dd, J = 9.7, 14.0, 1H), 2.46–2.40 (t, J = 8.1,

2H), 2.02–1.70 (m, 4H), 1.14–1.01 (t, J = 7.5, 3H); ES-

MS: mass calcd for C₂₈H₃₅N₆O₄519.3 (MH⁺). Found

m/z 519.5.

4.3.7. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(indol-3-yl)-

propan-2-yl-amide]-4-phenylbutan-2-yl-amide}semicarb-

azide (9). Following the procedure for 3 with the appro-

priate starting materials. White solid. Yield: 14 mg

(28%); HPLC (1): t_R= 4.03 min (>98%); HPLC (2):

1

t_R= 7.11 min (>99%); H NMR (CD₃OD, 250 MHz):

d 7.74–7.55 (d, J = 7.0, 1H), 7.33–7.00 (m, 8H), 4.79–

4.70 (m, 1H), 4.17–4.12 (dd, J = 5.3, 8.2, 1H), 3.88–

3.83 (t, J = 6.6, 1H), 3.43–3.32 (m, 1H, partially hidden

under methanol signal), 3.22–3.12 (dd, J = 8.6, 14.7,

1H), 2.58–2.49 (t, J = 8.5, 2H), 2.05–1.69 (m, 4H),

1.14–1.08 (t, J = 7.5, 3H); ES-MS: mass calcd for

C₂₆H₃₄N₇O₄508.3 (MH⁺). Found m/z 508.5.

4.3.12. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(m-meth-

oxyphenyl)propan-2-yl-amide]-3-(p-chlorophenyl)propan-

2-yl-amide}semicarbazide (14). Following the procedure

for 3 with the appropriate starting materials. White so-

lid. Yield: 12 mg (23%); HPLC (1): t_R= 4.14 min

(>96%); HPLC (2): t_R= 7.31 min (>97%); ¹H NMR

(CD₃OD, 250 MHz; one signal hidden under water

peak): d 7.41–7.18 (m, 5H), 6.98–6.89 (m, 4H), 4.78–

4.70 (m, 1H), 4.62–4.44 (dd, J = 5.6, 7.8, 1H), 3.33–

4.3.8. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(p-chloro-

phenyl)propan-2-yl-amide]-4-phenylbutan-2-yl-amide}semi-

carbazide (10). Following the procedure for 3 with the

appropriate starting materials. White solid. Yield:

4418

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

2.91 (m, 4H), 2.17–1.88 (m, 2H), 1.29–1.18 (t, J = 7.6,

3H); ES-MS: mass calcd for C₂₄H₃₂ClN₆O₅519.2

(MH⁺). Found m/z 519.4.

4.5. N^a-Boc-L-alaninehydrazide

CDI (4.71 g, 29.1 mmol) was added slowly to a solution

of N^a-Boc-L-alanine (5.0 g, 26.4 mmol) in 50 mL dry

THF. After addition the solution was stirred for

30 min. Then, hydrazine monohydrate (3.85 mL,

79.3 mmol) was added and the resulting solution was

stirred overnight. The reaction mixture was concentrated

and the white solid was dissolved in 1,4-dioxane and

lyophilized overnight. The crude product was suspended

in diethyl ether, cooled to ꢀ18 ꢂC and ﬁltered. The ﬁl-

trate was dried in vacuo overnight yielding 5.6 g of crude

product. The solid was a mixture of imidazole and the

desired product (imidazole–product 2:1, determined by

¹H NMR) and was used without further puriﬁcation.

4.3.13. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(1,1⁰-bi-

phenyl-4-yl)propan-2-yl-amide]-3-(p-hydroxyphenyl)propan-

2-yl-amide}semicarbazide (15). Following the procedure

for 3 with the appropriate starting materials. White so-

lid. Yield: 27 mg (49%); HPLC (1): t_R= 4.16 min

(>98%); HPLC (2): t_R= 7.06 min (>99%); ¹H NMR

(CD₃OD, 250 MHz): d 7.50–7.40 (m, 4H), 7.34–7.16

(m, 5H), 6.89–6.86 (d, J = 8.5, 2H), 6.60–6.54 (d,

J = 8.5, 2H), 4.56–4.50 (dd, J = 5.5, 8.6, 1H), 4.28–4.23

(dd, J = 5.9, 7.7, 1H), 3.70–3.64 (app t, J = 6.6, 1H),

3.16–3.08 (dd, J = 5.6, 13.9, 1H), 2.92–2.78 (m, 2H),

2.70–2.61 (dd, J = 7.8, 14.0, 1H), 1.90–1.69 (m, 2H),

1.02–0.86 (t, J = 7.4, 3H); ES-MS: mass calcd for

C₂₉H₃₅N₆O₅547.3 (MH⁺). Found m/z 547.5.

1

Yield (estimated): 3.3 g (62%). H NMR (DMSO-d₆ex-

changed with D₂O, 250 MHz): d 3.96–3.85 (m, 1H),

1.39–1.31 (s, 6H), 1.18–1.10 (d, J = 7.2, 3H).

4.3.14. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(m-ﬂuoro-

phenyl)propan-2-yl-amide]-3-(indol-3-yl)propan-2-yl-amide}-

semicarbazide (16). Following the procedure for 3 with

the appropriate starting materials. White solid. Yield:

13 mg (25%); HPLC (1): t_R= 3.97 min (>99%); HPLC

(2): t_R= 7.02 min (>99%); ¹H NMR (CD₃OD,

250 MHz): d 7.73–7.70 (d, J = 7.3, 1H), 7.49–7.03 (m,

8H), 4.75–4.68 (m, 1H), 4.63–4.58 (t, J = 7.0, 1H),

3.93–3.88 (m, 1H), 3.35–3.17 (m, 3H), 3.04–2.95 (dd,

J = 8.4, 13.8, 1H), 2.11–1.92 (m, 2H), 1.23–1.15 (t,

J = 7.5, 3H); ES-MS: mass calcd for C₂₅H₃₁FN₇O₄

512.3 (MH⁺). Found m/z 512.4.

4.6. N^a-Boc-L-aminobutyric acid hydrazide

CDI (1.76 g, 10.8 mmol) was added slowly to a solution

of N^a-Boc-L-aminobutyric acid (2.0 g, 9.84 mmol) in

20 mL dry THF. After addition the solution was stirred

for 1 h. Then, hydrazine monohydrate (1.44 mL,

29.5 mmol) was added and the resulting solution was

stirred overnight. The reaction mixture was concen-

trated and the white solid re-dissolved in 1,4-dioxane

and lyophilized overnight yielding 3.85 g of crude prod-

uct. The solid was a mixture of imidazole and desired

product (imidazole–product 2:1, determined by ¹H

NMR) and was used without further puriﬁcation. Yield

(estimated): 2.14 g (100%). ¹H NMR (DMSO-d₆

250 MHz): d 9.08–8.89 (s, 1H), 6.75–6.10 (d, J = 8.2,

1H), 3.79–3.64 (q, J = 7.91, 14.4, 1H), 1.61–1.34 (m,

2H), 1.34–1.18 (s, 9H), 0.81–0.63 (t, J = 7.54, 3H).

4.3.15. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(m-ﬂuoro-

phenyl)propan-2-yl-amide]-3-(2-naphthyl)propan-2-yl-amide}-

semicarbazide (17). Following the procedure for 3 with

the appropriate starting materials. White solid. Yield:

8 mg (15%); HPLC (1): t_R= 4.39 min (>96%); HPLC

(2): t_R= 7.44 min (>98%); ¹H NMR (CD₃OD,

250 MHz): d 7.89–7.68 (m, 4H), 7.50–7.24 (m, 4H),

7.16–6.92 (m, 3H), 4.68–4.62 (dd, J = 5.7, 8.8, 1H), 4.58–

4.53 (dd, J = 5.4, 8.1, 1H), 3.81–3.76 (app t, J = 6.6,

1H), 3.28–3.17 (m, 2H), 3.12–2.92 (m, 2H), 2.02–1.78

(m, 2H), 1.08–1.02 (t, J = 7.5, 3H); ES-MS: mass calcd

for C₂₇H₃₂FN₆O₄523.2 (MH⁺). Found m/z 523.4.

4.7. General procedure

CDI (1.1 equiv) was added to a solution of either N^a-

Boc-^L-alaninehydrazide or N^a-Boc-L-aminobutyric acid

hydrazide (1 equiv) in dry THF (1 mL/0.24 mmol hydr-

azide). The resulting solution was stirred for minimum

2 h and the appropriate amine (1.1 equiv) dissolved in

dry THF (0.5 mL/0.27 mmol amine) was added. The

reaction mixture was stirred overnight and then concen-

trated. The residue was treated with 95% TFA/H₂O

(1 mL/0.24 mmol hydrazide) for 5 min and concentrated

followed by HPLC puriﬁcation.

4.3.16. 1-(2S-2-Aminobutanoyl)-4-{2S-N-[2S-3-(m-ﬂuoro-

phenyl)propan-2-yl-amide]-3-(1,1⁰-biphenyl-4-yl)propan-2-

yl-amide}semicarbazide (18). Following the procedure

for 3 with the appropriate starting materials. White solid.

Yield: 5 mg (9%); HPLC (1): t_R₁= 4.68 min (>99%);

HPLC (2): t_R= 7.89 min (>98%); H NMR (CD₃OD,

250 MHz): d 7.65–6.92 (m, 13H), 4.68–4.63 (dd,

J = 5.7, 8.7, 1H), 4.51–4.46 (dd, J = 5.7, 7.9, 1H), 3.82–

3.77 (app t, J = 6.6, 1H), 3.25–2.88 (m, 4H), 1.99–1.87

(m, 2H), 1.15–1.02 (t, J = 7.5, 3H); ES-MS: mass calcd

for C₂₉H₃₄FN₆O₄549.3 (MH⁺). Found m/z 549.4.

4.7.1. 1-(2S-2-Aminopropionyl)-4-benzylsemicarbazide (19).

Yield: 23 mg (36%); HPLC (1): t_R= 2.57 min (>99%);

HPLC (2): t_R= 4.97 min (95%); ¹H NMR (D₂O,

250 MHz): d (rotamers observed) 7.40–7.23 (m, 5H),

4.32 (s, 2H), 4.15–4.05 (q, J = 7.2, 1H), 1.55–1.49 (d,

J = 7.2, 3H); ES-MS: mass calcd for C₁₁H₁₇N₄O₂

237.1 (MH⁺). Found m/z 237.

4.4. Synthesis of inhibitors 19–44

4.7.2. 1-(2S-2-Aminopropionyl)-4-(o-chlorobenzyl)semi-

carbazide (20). Yield: 24 mg (33%); HPLC (1):

t_R= 3.02 min (>99%); ¹H NMR (D₂O, 250 MHz): d

(rotamers observed) 7.23–7.01 (m, 4H), 4.18 (s, 2H),

3.94–3.84 (q, J = 7.2, 1H), 1.35–1.27 (d, J = 7.2, 3H);

The compounds were prepared starting from the Boc-

protected amino acid hydrazide derivatives via activa-

tion with CDI, followed by displacement with the

appropriate amine, as described below.

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

4419

ES-MS: mass calcd for C₁₁H₁₆ClN₄O₂271.1 (MH⁺).

Found m/z 271.0.

4.7.10. 1-(2S-2-Aminobutanoyl)-4-(o-bromobenzyl)semi-

carbazide (28). Yield: 49 mg (65%); HPLC (1):

t_R= 3.12 min (>99%); HPLC (2): t_R= 5.99 min

(>99%); ¹H NMR (CD₃OD, 250 MHz): d 7.57–7.53

(m, 1H), 7.43–7.28 (m, 2H), 7.19–7.12 (m, 1H), 4.42 (s,

2H), 3.86–3.81 (t, J = 6.6, 1H), 2.03–1.86 (m, 2H),

1.09–1.03 (t, J = 7.5, 3H); ES-MS: mass calcd for

C₁₂H₁₈BrN₄O₂329.1 (MH⁺). Found m/z 329.1.

4.7.3. 1-(2S-2-Aminopropionyl)-4-(m-chlorobenzyl)semi-

carbazide (21). Yield: 47 mg (70%); HPLC (1):

t_R= 3.17 min (>98%); ¹H NMR (D₂O, 250 MHz): d

(rotamers observed) 7.31–7.21 (m, 3H), 7.18–7.11 (m,

1H), 4.29 (s, 2H), 4.16–4.05 (m, J = 7.2, 1H), 1.61–1.53

(d, J = 7.2, 3H); ES-MS: mass calcd for C₁₁H₁₆ClN₄O₂

271.1 (MH⁺). Found m/z 271.1.

4.7.11.

1-(2S-2-Aminobutanoyl)-4-(o-nitrobenzyl)semi-

carbazide (29). Yield: 26 mg (37%); HPLC (1):

t_R= 2.80 min (>96%); HPLC (2): t_R= 5.34 min

(>99%); ¹H NMR (CD₃OD, 250 MHz): d 8.06–8.03

(m, 1H), 7.68–7.66 (m, 2H), 7.54–7.45 (m, 1H), 4.67 (s,

2H), 3.85–3.80 (t, J = 6.6, 1H), 1.99–1.86 (m, 2H),

1.09–1.03 (t, J = 7.5, 3H); ES-MS: mass calcd for

C₁₂H₁₈N₅O₄296.1 (MH⁺). Found m/z 296.3.

4.7.4. 1-(2S-2-Aminopropionyl)-4-(p-chlorobenzyl)semi-

carbazide (22). Yield: 52 mg (71%); HPLC (1):

t_R= 3.30 min (>97%); ¹H NMR (D₂O, 250 MHz): d

(rotamers observed) 7.37–7.30 (m, 2H), 7.27–7.19 (m,

2H), 4.29 (s, 2H), 4.16–4.05 (q, J = 7.2, 1H), 1.55–1.49

(d, J = 7.2, 3H); ES-MS: mass calcd for C₁₁H₁₆ClN₄O₂

271.1 (MH⁺). Found m/z 271.1.

4.7.12. 1-(2S-2-Aminobutanoyl)-4-(o-aminobenzyl)semi-

carbazide (30). This compound was an intermediate in

the synthesis of S44–S57 (Table S3) and was unstable

to storage. Yield: 15 mg (69%); ES-MS: mass calcd for

C₁₂H₂₀N₅O₂266.2 (MH⁺). Found m/z 266.4.

4.7.5. 1-(2S-2-Aminopropionyl)-4-(2-phenylethyl)semicar-

bazide (23). Yield: 45 mg (66%); HPLC (1): t_R=

3.11 min (>99%); HPLC (2): t_R= 5.35 min (96%);

¹H NMR (D₂O, 250 MHz): d (rotamers observed)

7.37–7.21 (m, 5H), 4.10–4.02 (q, J = 7.2, 1H), 3.40–

3.31 (td, J = 6.9, 2.5, 2H), 2.79–2.72 (t, J = 6.9, 2H),

1.50–1.45 (d, J = 7.2, 3H); ES-MS: mass calcd for

C₁₂H₁₉N₄O₂251.1 (MH⁺). Found m/z 251.0.

4.7.13. 1-(2S-2-Aminobutanoyl)-4-(o-methylthiobenzyl)-

semicarbazide (31). Yield: 20 mg (29%); HPLC (1):

t_R= 3.27 min (99>%); HPLC (2): t_R= 5.91 min

(>99%); ¹H NMR (CD₃OD, 250 MHz): d (rotamers ob-

served) 7.34–7.22 (m, 3H), 7.17–7.10 (m, 1H), 4.42 (s,

2H), 3.84–3.79 (t, J = 6.5, 1H), 1.99–1.86 (m, 2H),

1.09–1.03 (t, J = 7.4, 3H); ES-MS: mass calcd for

C₁₃H₂₁N₄O₂S 297.1 (MH⁺). Found m/z 297.2.

4.7.6. 1-(2S-2-Aminopropionyl)-4-(1-naphthylmethyl)semi-

carbazide (24). Yield: 42 mg (55%); HPLC (1):

t_R= 3.50 min (>97%); HPLC (2): t_R= 6.34 min

(>99%); ¹H NMR (D₂O, 250 MHz): d (rotamers ob-

served) 8.03–7.81 (m, 3H), 7.63–7.40 (m, 4H), 4.77 (s,

2H), 4.13–4.03 (q, J = 7.2, 1H), 1.52–1.45 (d, J = 7.2,

3H); ES-MS: mass calcd for C₁₅H₁₉N₄O₂287.1

(MH⁺). Found m/z 287.0.

4.7.14. 1-(2S-2-Aminobutanoyl)-4-(o-ethoxybenzyl)semi-

carbazide (32). Yield: 28 mg (41%); HPLC (1):

t_R= 3.27 min (>99%); HPLC (2): t_R= 6.10 min

(>98%); ¹H NMR (CD₃OD, 250 MHz): d (rotamers ob-

served) 7.27–7.17 (m, 2H), 6.93–6.83 (m, 2H), 4.36 (s,

2H), 4.12–4.03 (q, J = 6.95, 2H), 3.84–3.79 (t, J = 6.5,

1H), 2.01–1.80 (m, 2H), 1.46–1.40 (t, J = 7.0, 3H),

1.09–1.03 (t, J = 7.6, 3H); ES-MS: mass calcd for

C₁₄H₂₃N₄O₃295.2 (MH⁺). Found m/z 295.2.

4.7.7. 1-(2S-2-Aminobutanoyl)-4-benzylsemicarbazide (25).

Yield: 34.8 mg (60%); HPLC (1): t_R= 2.56 min (>99%);

HPLC (2): t_R= 4.82 min (96%); ¹H NMR (CD₃OD,

250 MHz): d (rotamers observed) 7.31–7.21 (m, 5H),

4.36 (s, 2H), 3.85–3.80 (t, J = 6.6, 1H), 1.99–1.86 (m,

2H), 1.09–1.03 (t, J = 7.5, 3H); ES-MS: mass calcd for

C₁₂H₁₉N₄O₂251.1 (MH⁺). Found m/z 251.3.

4.7.15. 1-(2S-2-Aminobutanoyl)-4-[o-(N-morpholinyl)benzyl]-

semicarbazide (33). Yield: 32 mg (41%); HPLC (1):

t_R= 2.55 min (>99%); HPLC (2): t_R= 4.75 min (97%); H

NMR (CD₃OD, 250 MHz): d (rotamers observed) 7.52–

7.30 (m, 4H), 4.49 (s, 2H), 4.03–3.99 (m, 4H), 3.87–3.82

(t, J = 6.6, 1H), 3.39–3.30 (m, 4H), 2.03–1.86 (m, 2H),

1.10–1.04 (t, J = 7.5, 3H); ES-MS: mass calcd for

C₁₆H₂₆N₅O₃336.2 (MH⁺). Found m/z 336.3.

4.7.8. 1-(2S-2-Aminobutanoyl)-4-(o-chlorobenzyl)semi-

carbazide (26). Yield: 9.4 mg (13%); HPLC (1):

t_R= 3.39 min (>99%); HPLC (2): t_R= 5.76 min

(>99%); ¹H NMR (CD₃OD, 250 MHz): d (rotamers ob-

served) 7.41–7.28 (m, 2H), 7.26–7.13 (m, 2H), 4.41 (s,

2H), 3.80–3.71 (t, J = 6.6, 1H), 1.96–1.79 (m, 2H),

1.07–0.95 (t, J = 7.5, 3H); ES-MS: mass calcd for

C₁₂H₁₈ClN₄O₂285.1 (MH⁺). Found m/z 285.1.

1

4.7.9. 1-(2S-2-Aminobutanoyl)-4-methyl-4-(o-chlorobenz-

yl)semicarbazide (27). Yield: 52 mg (75%); HPLC (1):

t_R= 3.28 min (>99%); HPLC (2): t_R= 6.15 min

(>99%); ¹H NMR (CD₃OD, 250 MHz): d (rotamers ob-

served) 7.42–7.37 (m, 1H), 7.32–7.23 (m, 3H), 4.65 (s,

2H), 3.87–3.81 (t, J = 6.6, 1H), 2.96 (s, 3H), 2.00–1.87

(m, 2H), 1.11–1.05 (t, J = 7.5, 3H); ES-MS: mass calcd

for C₁₃H₂₀ClN₄O₂299.1 (MH⁺). Found m/z 299.3.

4.7.16. 1-(2S-2-Aminobutanoyl)-4-[o-(N-piperidinyl)benz-

yl]semicarbazide (34). Yield: 7 mg (9%); HPLC (1):

t_R= 2.71 min (>96%); HPLC (2): t_R= 4.75 min (95%);

¹H NMR (CD₃OD, 250 MHz): d (rotamers observed)

7.77–7.52 (m, 4H), 4.43 (s, 2H), 3.87–3.82 (t, J = 6.5,

1H), 3.70–3.57 (m, 4H), 2.21–2.02 (m, 4H), 1.99–1.71

(m, 4H), 1.11–1.05 (t, J = 7.4, 3H); ES-MS: mass calcd

for C₁₇H₂₈N₅O₂334.2 (MH⁺). Found m/z 334.2.

4420

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

1

3.53 min (>99%); HPLC (2): t_R= 6.49 min (>95%); H

4.7.17. 1-(2S-2-Aminobutanoyl)-4-[(1,1⁰-biphenyl-2-yl)-

methyl]semicarbazide (35). Yield: 63 mg (83%); HPLC:

t_R= 3.97 min (>97%); H NMR (CD₃OD, 250 MHz):

d (rotamers observed) 7.50–7.19 (m, 9H), 4.28 (s, 2H),

3.82–3.77 (t, J = 6.6, 1H), 2.01–1.85 (m, 2H), 1.09–1.03

(t, J = 7.4, 3H); ES-MS: mass calcd for C₁₈H₂₃N₄O₂

327.2 (MH⁺). Found m/z 327.4.

NMR (CD₃OD, 250 MHz): d (rotamers observed)

8.10–8.06 (m, 1H), 7.90–7.78 (m, 2H), 7.54–7.39 (m,

4H), 4.84 (partially hidden under H₂O, 2H), 3.85–3.79

(t, J = 6.6, 1H), 1.99–1.86 (m, 2H), 1.09–1.02 (t,

J = 7.5, 3H); ES-MS: mass calcd for C₁₆H₂₁N₄O₂

301.2 (MH⁺). Found m/z 301.3.

1

4.7.18. 1-(2S-2-Aminobutanoyl)-4-[o-(o-hydroxymethyl-

phenylthio)benzyl]semicarbazide (36). Yield: 25 mg

(28%); HPLC (1): t_R= 3.66 min (>98%); HPLC (2):

4.7.25. 1-(2S-2-Aminohexanoyl)-4-(o-chlorobenzyl)semi-

carbazide (43). CDI (45 mg, 0.28 mmol) was added

slowly to a solution of N^a-Boc-L-aminocaproic acid

(58 mg, 0.25 mmol) in 1.0 mL dry THF. After addition

the solution was stirred for 2 h. Then, hydrazine mono-

hydrate (13.4 lL, 0.28 mmol) was added and the result-

ing solution was stirred overnight. CDI (45 mg,

0.28 mmol) was added and the solution was stirred for

6 h. o-Chlorobenzylamine (27 lL, 0.23 mmol) was

added and the reaction mixture was stirred overnight,

and concentrated. The residue was treated with 1 mL

95% TFA/H₂O for 2 h, and concentrated followed by

HPLC puriﬁcation. Yield: 30 mg (38%); HPLC (1):

t_R= 3.46 min (>99%); HPLC (2): t_R= 6.44 min (97%);

¹H NMR (CD₃OD, 250 MHz): d (rotamers observed)

7.44–7.33 (m, 2H), 7.31–7.21 (m, 2H), 4.46 (s, 2H),

3.88–3.82 (t, J = 6.9, 1H), 1.96–1.79 (m, 2H), 1.47–1.36

(m, 4H), 0.99–0.92 (t, J = 7.22, 3H); ES-MS: mass calcd

for C₁₄H₂₂ClN₄O₂313.1 (MH⁺). Found m/z 313.1.

1

t_R= 6.63 min (>98%); H NMR (CD₃OD, 250 MHz):

d (rotamers observed) 7.57–7.07 (m, 8H), 4.72 (s, 2H),

4.46 (s, 2H), 3.85–3.79 (t, J = 6.6, 1H), 1.99–1.86 (m,

2H), 1.09–1.03 (t, J = 7.4, 3H); ES-MS: mass calcd for

C₁₉H₂₅N₄O₃S 389.2 (MH⁺). Found m/z 389.1.

4.7.19. 1-(2S-2-Aminobutanoyl)-4-[(1,1⁰-biphenyl-4-yl)-

methyl]semicarbazide (37). Yield: 34 mg (45%); HPLC

(1): t_R= 3.86 min (>99%); ¹H NMR (CD₃OD,

250 MHz): d (rotamers observed) 7.60–7.55 (m, 4H),

7.45–7.29 (m, 5H), 4.41 (s, 2H), 3.86–3.81 (t, J = 6.5,

1H), 2.00–1.87 (m, 2H), 1.10–1.04 (t, J = 7.4, 3H); ES-

MS: mass calcd for C₁₈H₂₃N₄O₂327.2 (MH⁺). Found

m/z 327.2.

4.7.20. 1-(2S-2-Aminobutanoyl)-4-[p-(thiophen-2-yl)benz-

yl]semicarbazide (38). Yield: 29 mg (37%); HPLC (1):

t_R= 3.92 min (>99%); HPLC (2): t_R= 6.79 min (98%);

¹H NMR (CD₃OD, 250 MHz): d (rotamers observed)

7.60–7.54 (m, 2H), 7.35–7.31 (m, 4H), 7.08–7.05 (m,

1H), 4.38 (s, 2H), 3.85–3.80 (t, J = 6.6, 1H), 2.00–1.86

(m, 2H), 1.10–1.04 (t, J = 7.5, 3H); ES-MS: mass calcd

for C₁₆H₂₁N₄O₂S 333.1 (MH⁺). Found m/z 333.2.

4.7.26. 1-(2S-2-Amino-3-phenyl-propionyl)-4-(o-chloro-

benzyl)semicarbazide (44). CDI (45 mg, 0.28 mmol) was

added slowly to a solution of N^a-Boc-L-phenylalanine

(67 mg, 0.25 mmol) in 1.0 mL dry THF. After addition

the solution was stirred for 2 h. Then, hydrazine mono-

hydrate (13.4 lL, 0.28 mmol) was added and the result-

ing solution was stirred overnight. CDI (45 mg,

0.28 mmol) was added and the solution was stirred for

6 h. 2-Chlorobenzylamine (27 lL, 0.23 mmol) was

added and the reaction mixture was stirred overnight,

and concentrated. The residue was treated with 1 mL

95% TFA/H₂O for 2 h, and concentrated followed by

HPLC puriﬁcation. Yield: 15.6 mg (18%); HPLC (1):

t_R= 3.71 min (>97%); HPLC (2): t_R= 6.85 min (95%);

¹H NMR (CD₃OD, 250 MHz): d (rotamerism observed)

7.38–7.14 (m, 9H), 4.41 (s, 2H), 3.80–3.61 (m, 3H); ES-

MS: mass calcd for C₁₇H₂₀ClN₄O₂347.1 (MH⁺). Found

m/z 347.1.

4.7.21. 1-(2S-2-Aminobutanoyl)-4-(2-chloro-6-ﬂuorobenz-

yl)semicarbazide (39). Yield: 14 mg (20%); HPLC (1):

t_R= 3.03 min (>99%); HPLC (2): t_R= 5.69 min (98%);

¹H NMR (CD₃OD, 250 MHz): d (rotamers observed)

7.37–7.24 (m, 2H), 7.13–7.06 (m, 1H), 4.56 (s, 2H),

3.81–3.76 (t, J = 6.5, 1H), 1.98–1.84 (m, 2H), 1.09–1.03

(t, J = 7.6, 3H); ES-MS: mass calcd for C₁₂H₁₇ClFN₄O₂

303.1 (MH⁺). Found m/z 303.0.

4.7.22. 1-(2S-2-Aminobutanoyl)-4-(2,6-diﬂuorobenzyl)-

semicarbazide (40). Yield: 13 mg (19%); HPLC (1):

t_R= 2.74 min (>99%); ¹H NMR (D₂O, 250 MHz): d

(rotamers observed) 7.44–7.32 (m, 1H), 7.08–6.97 (m,

2H), 4.45 (s, 2H), 4.04–3.99 (t, J = 6.6, 1H), 2.02–1.90

(m, 2H), 1.06–1.00 (t, J = 7.5, 3H); ES-MS: mass calcd

for C₁₂H₁₇F₂N₄O₂287.1 (MH⁺). Found m/z 287.2.

4.7.27. 1-(2S-2-Aminobutanoyl)-2-benzyl-4-(o-chlorobenz-

yl)semicarbazide (45). (a) Preparation of 1-Fmoc-2-

benzylhydrazine (I, following a procedure for 1-Fmoc-

2-methylhydrazine³¹): Benzylhydrazine dihydrochloride

(2 g, 10.25 mmol) and DIPEA (3.5 mL, 20.5 mmol) were

dissolved in dry CHCl₃(10 mL). This solution was

added in a single portion to a stirring solution of Boc₂O

in dry CHCl₃(10 mL) at ꢀ78 ꢂC. The reaction was al-

lowed to come to rt and then stirred for 2 h. Fmoc–Cl

(2.7 g, 10.25 mmol) was then added in a single portion

and DIPEA (1.8 mL, 10.25 mmol) was added dropwise.

The resulting solution was stirred at rt for 6 h and then

TFA–H₂O 95:5 (15 mL) was added carefully. After 2 h

the bright yellow solution was taken to dryness. The resi-

due was redissolved in EtOAc (100 mL), washed care-

fully with satd NaHCO₃(·3) and brine (·1) and dried

4.7.23. 1-(2S-2-Aminobutanoyl)-4-(2,4-dichlorobenzyl)-

semicarbazide (41). Yield: 26 mg (36%); HPLC (1):

t_R= 3.50 min (>99%); HPLC (2): t_R= 6.51 min (95%);

¹H NMR (D₂O, 250 MHz): d (rotamers observed)

7.51–7.50 (m, 1H), 7.34–7.32 (m, 2H), 4.41 (s, 2H),

4.07–4.01 (t, J = 6.6, 1H), 2.04–1.92 (m, 2H), 1.07–1.01

(t, J = 7.6, 3H); ES-MS: mass calcd for C₁₂H₁₇Cl₂N₄O₂

319.1 (MH⁺). Found m/z 319.1.

4.7.24. 1-(2S-2-Aminobutanoyl)-4-(1-naphthylmethyl)semi-

carbazide (42). Yield: 32 mg (46%); HPLC (1): t_R=

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

4421

(MgSO₄). Evaporation gave a yellowish solid, which was

puriﬁed on silica, using PE–EtOAc 2:1. The resulting

oﬀ-white solid (3 g) was dissolved in hot EtOAc

(20 mL), then cooled slowly to rt and ﬁnally kept at

5 ꢂC overnight. The precipitate was washed with cold

EtOAc and dried to give a white solid. Yield 1.2 g

(34%). The ﬁltrate was concentrated to dryness and then

heated brieﬂy in DE (20 mL). Upon cooling, wash with

DE and drying a further 0.4 g white solid could be iso-

lated having the same purity as the ﬁrst crop. Combined

yield: 1.6 g (46%). HPLC (1): t_R= 5.39 (>98%); ¹H

NMR (DMSO-d₆250 MHz): d 8.73 (br s, 1H), 7.90–

7.87 (d, J = 7.4, 2H), 7.71–7.68 (d, J = 7.3, 2H), 7.45–

7.21 (m, 9 H), 4.95 (br s, 1H), 4.35–4.33 (d, J = 6.5,

2H), 4.25–4.19 (t, J = 6.5, 1H), 3.91 (s, 2H); ¹³C NMR

(DMSO-d₆250 MHz): d 156.8, 143.7, 140.7, 138.6,

128.4, 128.0, 127.6, 127.0, 126.8, 125.2, 120.0, 65. 4,

54.2, 46.7; ES-MS: mass calcd for C₂₂H₂₁N₂O₂345.15

(MH⁺). Found m/z 345.21.

washed once with satd aq NaHCO₃, 10% aq citric acid

and brine. Drying over MgSO₄followed by evaporation

gave an oil, which was dried in vacuo to give a white so-

lid. Yield: 6.75 g (96%); HPLC (1): t_R= 5.00 (>99%); ¹H

NMR (CDCl₃, 250 MHz): d 9.00 (br, 1H), 7.29 (m, 5H),

7.27 (br, 1H), 6.15–6.11 (d, J = 8.4, 1H), 5.12–4.98 (m,

2H), 4.45–4.40 (m, 1H), 2.57–2.52 (t, J = 7.1, 2H),

2.13–1.89 (m, 5H), 1.42 (s, 9H); ¹³C NMR (CDCl₃,

250 MHz): d 171.7, 156.7, 155.7, 136.5, 128.9, 128.5,

128.4, 81.9, 67.5, 52.8, 32.3, 30.3, 28.5, 15.6; ES-MS:

mass calcd for C₁₈H₂₈N₃O₅S 398.2 (MH⁺). Found m/z

398.1.

(b) Preparation of Cbz-^L-Met-NHNHBoc methyl sulfo-

nium iodide (III): Cbz-^L-Met-NHNHBoc (6.65 g,

16.73 mmol) was stirred in methyl iodide (25 mL) at rt

for a period of 72 h. DCM (50 mL) was added and the

yellow solution was concentrated to give a yellow oil,

which was dried in vacuo to give the sulfonium salt as

a

yellow solid. Yield: 9.1 g (100%); HPLC (1):

1

(b) Preparation of 1-(2S-2-Aminobutanoyl)-2-benzyl-4-

(o-chlorobenzyl)semicarbazide (45): 1-Fmoc-2-ben-

zylhydrazine (I, 100 mg, 0.29 mmol) was dissolved in

dry THF (1 mL). Phosgene (1.9 M solution in toluene,

300 lL, 0.58 mmol) was added at 0 ꢂC and the reaction

mixture was then cooled to rt and stirred for 16 h. Evap-

oration to dryness gave crude 2-(chlorocarbonyl)-1-

Fmoc-2-benzylhydrazine as a clear oil. ES-MS: mass

calcd for C₂₃H₁₉ClN₂O₃Na 429.11 (MNa⁺). Found m/z

429.1. The crude product was dissolved in dry CH₃CN

(1 mL) and DIPEA (50 lL, 0.29 mmol) and 2-chloro-

benzylamine (39 lL, 0.32 mmol) was added. After

10 min the reaction mixture had turned nearly solid

and a further 1.5 mL of CH₃CN was added. After 3 h

LC–MS showed complete conversion to II (m/z 512.1;

MH⁺for C₃₀H₂₇ClN₃O₃). Piperidine (86 lL, 0.87 mmol)

was added and the resulting bluish-green solution was

stirred at rt overnight. The now brownish reaction mix-

ture was split into two equal portions. To one portion

was added (Boc-Abu)₂O (formed from Boc-Abu-OH

(177 mg, 0.87 mmol) and DIC (68 lL, 0.44 mmol) in

1 mL dry THF; reaction time 20 min). After 3 days the

mixture was treated with TFA–H₂O 95:5 (5 mL) for

1 h and evaporated to dryness. The dark residue was

puriﬁed by HPLC to give the title compound as a white

solid. Yield: 4.6 mg (9%); HPLC (1): t_R= 4.03 min

(>99%); HPLC (2): t_R= 6.92 min (>95%); ¹H NMR

(CD₃OD, 250 MHz): d 7.58–7.37 (m, 9H), 5.15–5.00

(m, 1H; partially hidden under water signal), 4.74–4.60

(m, 3H), 3.93–3.88 (app t, 1H), 2.03–1.79 (m, 2H),

1.50–1.46 (t, J = 7.5, 3H); ES-MS: mass calcd for

C₁₉H₂₃ClN₄O₂375.2 (MH⁺). Found m/z 375.2.

t_R= 3.86 (>99%); H NMR (CDCl₃, 250 MHz): d 9.33

(br, 1H), 7.27–7.19 (m, 5H), 6.93 (br, 1H), 6.38–6.36

(d, J = 7.1, 1H), 5.01 (s, 2H), 4.61–4.59 (m, 1H), 3.75–

3.58 (m, 2H), 3.08 (s, 3H), 2.99 (s, 3H), 2.45–2.30 (m,

2H), 1.34 (s, 9H); ¹³C NMR (CDCl₃, 250 MHz): d

171.1, 156.9, 155.8, 136.5, 129.0, 128.6, 128.4, 82.0,

67.7, 52.3, 40.3, 28.7, 26.8, 26.4; ES-MS: mass calcd

for C₁₉H₃₀N₃O₅S 412.2 (M⁺). Found m/z 412.1.

(c) Preparation of 3(S)-benzyloxycarbonylamino-1-tert-

butoxycarbonylaminopyrrolidin-2-one (IV): Cbz-^L-

Met-NHNHBoc methyl sulfonium iodide (III, 9 g,

16.68 mmol) was dissolved in dry DCM–DMF 1:1

(338 mL). The resulting yellow solution was cooled in

an ice bath under Ar and NaH (60% suspension in min-

eral oil; 1344 mg, 34 mmol) was added in one portion.

The solution was stirred at 0 ꢂC for 3 h, during which

time the colour changed from yellow to various shades

of red. The reaction was quenched by addition of EtOAc

(115 mL) and water (27 mL), and then stirred overnight

at room temperature. The volume was concentrated to

ca. 100 mL, diluted with water (300 mL) and extracted

with EtOAc (·3). The combined organics were washed

with brine, dried over MgSO₄and concentrated to give

the crude product as a semisolid residue (6 g after dry-

ing). This was dissolved in hot EtOAc–DE 1:2

(60 mL), then cooled slowly to rt and ﬁnally to

ꢀ18 ꢂC. After 1 h a further 10 mL DE and 20 mL n-hep-

tane were added portionwise to the cold solution. Upon

storage at ꢀ18 ꢂC overnight a white solid was isolated,

washed with n-heptane (·3) and dried. Yield: 2.9 g

(50%); HPLC (1): t_R= 4.68 (>86%)/4.97 (>12%) (iso-

1

baric peaks); H NMR (CDCl₃, 250 MHz): d 7.28–7.19

4.7.28. 1,1-(2S-1,4-cyclo-Aminobutanoyl)-4-(o-chloroben-

zyl)semicarbazide (46). (a) Preparation of Cbz-^L-Met-

NHNHBoc: Cbz-^L-methionine (5 g, 17.65 mmol) was

dissolved in dry THF (20 mL) and cooled in an ice bath.

CDI (3.15 g, 19.42 mmol) was added in one portion,

resulting in vigorous evolution of gas. After 20 min

BocNHNH₂(2.57 g, 19.42 mmol) was added and the

resulting solution was stirred for a period of 20 h, at

which time it was poured into water (150 mL) and ex-

tracted with EtOAc (·3). The combined solvents were

(m, 5H), 6.81 (s, 1H), 5.51–5.49 (d, J = 7.1, 1H), 5.03

(s, 2H), 4.30–4.20 (m, 1H), 3.58–3.37 (m, 2H), 2.58–

2.47 (m, 1H), 2.02–1.70 (m, 1H), 1.39 (s, 9H); ¹³C

NMR (CDCl₃, 250 MHz): d 172.1, 156.8, 154.7, 136.7,

128.9, 128.5, 128.4, 82.2, 67.3, 51.2, 46.3, 28.5, 26.4;

ES-MS: mass calcd for C₁₇H₂₄N₃O₅350.2 (MH⁺).

Found m/z 350.1.

(d) Preparation of 3(S)-benzyloxycarbonylamino-1-

aminopyrrolidin-2-one (V): 3(S)-Benzyloxycarbonyl-

4422

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

amino-1-tert-butoxycarbonylaminopyrrolidin-2-one (IV,

100 mg, 0.286 mmol) was dissolved in HCl in dioxane

(4 M, 2 mL) and left at rt for 18 h. The solution was

evaporated to dryness and the crude residue was puriﬁed

by preparative HPLC to give a white solid. Yield: 35 mg

(49%); HPLC (1): t_R= 3.61 (>99%); ES-MS: mass calcd

for C₁₂H₁₆N₃O₃250.1 (MH⁺). Found m/z 250.1.

KCl, 1.5 mM EDTA and 5 mM cysteamine). The reac-

tion was initiated by the addition of 1 mU enzyme. Exci-

tation: 320 nm, emission: 420 nm.

K_iDetermination was performed using four diﬀerent

substrate concentrations (100, 150, 225 and 300 lM) in

the absence or presence of 8 at ﬁve diﬀerent concentra-

tions: 10, 25, 50, 100 and 150 nM.

(e) Preparation of 1,1-(2S-1,4-cyclo-benzyloxycarbonyl-

aminobutanoyl)-4-(o-chlorobenzyl)semicarbazide (VI):

3(S)-Benzyloxycarbonylamino-1-aminopyrrolidin-2-one

(V, 35 mg, 0.14 mmol) and NEM (35 lL, 0.28 mmol)

were dissolved in dry THF (1.5 mL) and CDI (25 mg,

0.154 mmol) was added. After a period of 21 h at rt o-

chlorobenzylamine (19 lL, 0.154 mmol) was added.

After 4.5 h the crude product was puriﬁed by prepara-

tive HPLC to give a white solid. Yield: 20 mg (34%);

HPLC (1): t_R= 4.84 (>99%); ES-MS: mass calcd for

C₂₀H₂₂N₄O₄417.1 (MH⁺). Found m/z 417.0.

Reversibility studies were performed using 150 nM of 8

and compared with the reaction in absence of inhibitor,

or in the presence of 20 lM of the irreversible inhibitor

GFCH₂N₂. After 6000 s, substrate was added to a con-

centration of 1.5 mM (10 · K_m).³³The reaction was fol-

lowed for additional 7000 s.

4.10. Human liver cathepsin B assay

The human liver cathepsin B (enzyme system products)

assay was performed in assay buﬀer (0.1 M MES buﬀer

pH 6.1, 1 mM EDTA, 5 mM DTT). Inhibitor and 1 ng

enzyme were incubated for 5 min at 37 ꢂC. The reaction

was initiated by addition of the following substrate:

Boc-Leu-Arg-Arg-AFC*2TFA (Enzyme System Prod-

ucts) to 600 lM. Excitation: 400 nm, emission: 505 nm.

(f) Preparation of 1,1-(2S-1,4-cyclo-aminobutanoyl)-4-

(o-chlorobenzyl)semicarbazide (46): 1,1-(2S-1,4-cyclo-

benzyloxycarbonylaminobutanoyl)-4-(o-chlorobenzyl)-

semicarbazide (VI, 19 mg, 46 lmol) was dissolved in

glacial acetic acid (300 lL). HBr in acetic acid (5.7 M,

20 lL, 114 lmol) was added. After 22 h a further

20 lL HBr in acetic acid was added and yet another

20 lL after a further 3.5 h. Three hours later the crude

product was puriﬁed by preparative HPLC to give a

white solid. Yield: 10.7 mg (83%); HPLC (1): t_R= 3.34

(>99%); HPLC (2): t_R= 5.69 min (>99%); ¹H NMR

(CDCl₃, 250 MHz): d 7.56–7.34 (m, 4H), 4.59 (s, 2H),

4.29–4.21 (t, J = 9.1, 1H), 3.85–3.66 (m, 2H), 2.78–2.66

(m, 1H), 2.34–2.21 (m, 1H); ES-MS: mass calcd for

C₁₂H₁₆ClN₄O₂283.1 (MH⁺). Found m/z 283.1.

4.11. Human cathepsin G assay

Assay buﬀer (100 mM Tris/HCl pH 8.3, 500 mM NaCl,

20 mM CaCl₂, 125 lM DTNB), 20 ng human cathepsin

G (Calbiochem) and inhibitor were incubated for 10 min

at 37 ꢂC. The reaction was started by the addition of

substrate (Suc-Ala-Ala-Pro-Phe-SBzl, Bachem) to

200 lM. The absorbance was monitored at 410 nm.

4.12. Human liver cathepsin H assay

4.8. Enzymatic assays

Human liver cathepsin H (enzyme system products) was

pre-incubated in the following buﬀer: 50 mM sodium

phosphate buﬀer pH 6.0, 2 mM EDTA, 0.012% TX-

100, 5 mM DTT, for 5 min on ice. The ﬁnal assay was

performed with inhibitor, 40 ng enzyme and 15 lM sub-

strate (Ala-Arg-Gly-AFC*2HBr, enzyme system prod-

ucts). Excitation: 400 nm, emission: 505 nm.

All enzyme assays were performed at 37 ꢂC in duplicate

in 96-well plates. The reaction was monitored for

10 min. In the software (SOFTmax Pro) used for data

collection from the ﬂuorometer (Molecular Devices:

Gemini EM) or the spectrophotometer (Molecular De-

vices: SPECTRAmax 190), it was ensured that the mea-

sured control slopes were linear (R²> 0.99). For K_m

determinations, data were exported to GraphPad Prism

and ﬁtted to the Michaelis–Menten equation. For IC₅₀

determinations, data were exported to GraphPad Prism

and nonlinear regression were performed using the op-

tion Sigmoidal dose–response (variable slope). Data

for K_ideterminations were analyzed using the SigmaPlot

Enzyme Kinetics Module (SPSS Science).

4.13. Human liver cathepsin L assay

Human liver cathepsin L (enzyme system products) was

activated for 5 min on ice in assay buﬀer (20 mM so-

dium acetate buﬀer pH 5.5, 4 mM EDTA, 0.012% TX-

100, 5 mM DTT). The ﬁnal assay was performed with

inhibitor, 80 nU enzyme and 10 lM substrate (Z-Phe-

ARG-AFC*TFA, enzyme system products). Excitation:

400 nm, emission: 505 nm.

4.9. Recombinant human dipeptidyl peptidase I (DPPI)

assay

4.14. Aspergillus DPPIV assay

Human DPPI was expressed and puriﬁed as described

previously.⁷The enzyme was activated by incubation

with an equal volume of 200 mM cysteamine at 37 ꢂC

for 5 min. Inhibitor and 150 lM substrate (Gly-

Tyr(3⁰NO₂)-Gly-Pro-Pro-Lys(e-Abz)-Gly, Schafer-N,

Denmark) were incubated for 5 min at 37 ꢂC in assay

buﬀer (100 mM sodium phosphate pH 6.0, 150 mM

Aspergillus DPPIV (0.04 mU, enzyme system products)

was added to a 30 ꢂC pre-heated assay buﬀer (50 mM

Tris buﬀer pH 7.4, 0.1% Triton X-100) containing inhib-

itor. Substrate (Gly-Pro-AFC*TFA, enzyme system

products) was added to 100 lM. The reaction was moni-

tored at 30 ꢂC. Excitation: 400 nm, emission: 505 nm.

J. Bondebjerg et al. / Bioorg. Med. Chem. 13 (2005) 4408–4424

4423

4.15. Human neutrophil elastase assay

3000 rpm for 10 min. The resulting supernatant was

transferred to eppendorf tubes. The Coomassie Plus

Protein Assay (Pierece) was used to determine protein

levels of the samples.

Human neutrophil elastase (Calbiochem) activity was

measured in assay buﬀer (50 mM sodium acetate buﬀer

pH 5.5, 200 mM NaCl). The assay was performed with

inhibitor, 10 ng enzyme and 400 lM substrate (MeO-

Suc-Ala-Ala-Pro-Val-AMC, Calbiochem). Excitation:

380 nm, emission: 460 nm.

4.18.4. Measurement of cellular DPPI activity. The activ-

ity of DPPI in cell lysates was performed in 100 mM so-

dium phosphate, 150 mM KCl, 1.5 mM EDTA (pH = 6)

using 50 lM of Gly-Tyr(3⁰NO₂)-Gly-Pro-Pro-Lys(e-

Abz)-Gly (Schafer-N, Denmark) as a substrate. For

each measurement, cell lysate corresponding to 50 lg

of protein was used. All samples were prepared in

duplicates.

4.16. Tryptase assay

Recombinant human skin b-tryptase (Promega) was di-

luted in assay buﬀer (100 mM HEPES pH 7.5, 10% glyc-

erol, 0.1 mg/mL heparin). Inhibitor and 0.4 ng enzyme

were incubated for 10 min at 37 ꢂC. Substrate

(Tyr(3⁰NO₂)-Arg-Lys-Tyr-Arg-Met-Ala-Ser-Ala-Lys(e-

Abz), Schafer-N, Denmark) was added to 7.5 lM. Exci-

tation: 320 nm, emission: 420 nm.

Acknowledgments

We gratefully acknowledge Jannie Rosendahl Christen-

sen, Helle Fjordvang, Sanne Rasmussen and Iben Jonas-

sen for their technical support.

4.17. Cytochrome P450 assays

Vivid^ꢁCYP assay kits for CYP1A2, CYP2C19,

CYP2C9, CYP2D6 and CYP3A4 were purchased from

invitrogen. Assays were performed according to the

manufacturerꢁs instructions.

Supplementary data

Tables S1 (S1–S6), S2 (S7–S43) and S3 (S44–S57) and

experimental details for 1, S1–S6 and S44. Supplemen-

tary data associated with this article can be found, in

the online version, at doi:10.1016/j.bmc.2005.04.048.

4.18. Cellular assays

4.18.1. Cell culture. The RBL-2H3 (rat basophilic leu-

kaemia cell line) and HepG2 (human epithelial hepacel-

lular carcinoma) cells were obtained from the American

Type Culture Collection (ATCC). RBL-2H3 cells were

grown in RPMI Medium (GIBCO) and HepG2 cells

were grown in minimum essential medium (MEM) a

medium. Media for cell culture were supplemented with

10% heat-inactivated foetal bovine serum (FBS; GIB-

CO), 100 U/mL penicillin and 100 lg/mL streptomycin

(GIBCO). Cells were cultured at 37 ꢂC in humidiﬁed

air atmosphere with 5% CO₂.

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Article Doi

Novel semicarbazide-derived inhibitors of human dipeptidyl peptidase I (hDPPI)

DOI: 10.1016/j.bmc.2005.04.048

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Full text of DOI:10.1016/j.bmc.2005.04.048

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