Syntheses and Different Chemical Behaviour of Precursors of Putative Dibasic Inhibitors of Human Mast Cell Tryptase

Article abstract of DOI:10.1007/s00706-003-0029-x

Choosing the best conditions and pathways for the synthesis of peptidic compounds remains a challenge for the peptide chemist. Our efforts towards the syntheses of two precursors of potential tryptase inhibitors, building block A and B, led to the development of two different synthesis routes. Each of them is successful in the synthesis of only one of the two, structurally nearly identical target compounds.

Full text of DOI:10.1007/s00706-003-0029-x

Monatshefte f€ur Chemie 134, 1159–1166 (2003)
DOI 10.1007/s00706-003-0029-x
Syntheses and Different Chemical Behaviour
of Precursors of Putative Dibasic Inhibitors
of Human Mast Cell Tryptase
ꢀ
Gregor Radau
Institute of Pharmacy, Ernst-Moritz-Arndt-University, Pharmaceutical=Medicinal Chemistry,
D-17487 Greifswald, Germany
Received January 9, 2003; accepted January 21, 2003
Published online June 2, 2003 # Springer-Verlag 2003
Summary. Choosing the best conditions and pathways for the synthesis of peptidic compounds
remains a challenge for the peptide chemist. Our efforts towards the syntheses of two precursors of
potential tryptase inhibitors, building block A and B, led to the development of two different synthesis
routes. Each of them is successful in the synthesis of only one of the two, structurally nearly identical
target compounds.
Keywords. Peptide synthesis; Tryptase-inhibitor.
Introduction
Human mast cell tryptase (E.C. 3.4.21.59), a member of the trypsin-like serine
proteases family and one of the major mast cell secretory proteases, is thought to
play a central role in mast cell related pathologies. Elevated concentrations of
tryptase were measured in allergic and inflammatory diseases, including asthma
[1, 2], allergic conjunctivitis [3], allergic rhinitis [4], rheumatic arthritis [5], multi-
ple sclerosis [6], interstitial cystitis [7], or psoriasis [8]. Tryptase is generated
and stored as a catalytically active tetramer [9] and – as a result of mast cell
activation – tryptase is released together with histamine, heparines, and other
mast cell proteases. Inhibition of tryptase presents a notable measure for improv-
ing the treatment of the above mentioned disorders.
Results and Discussion
In the course of our continuing studies towards the synthesis of cyanopeptide-
derived inhibitors of trypsin-like serine proteases [10–13], we have synthesised
thrombin inhibitors (e.g. RA-1002, Fig. 1) using cyanopeptides like aeruginosin
ꢀ
Corresponding author. E-mail: radau@pharmazie.uni-greifswald.de

1160
G. Radau
Fig. 1. Development of putative tryptase inhibitors
98-B (isolated from Microcystis aeruginosa NIES-98) [14] as new lead structures.
Compound RA-1002, our first selective acting inhibitor of thrombin, was the start-
ing point for the development of putative tryptase inhibitors. The amino acid
sequence of RA-1002 was the base for molecular modeling studies on analogous
compounds. Since these studies suggested that RA-1003 and RA-1004 should be
able to inhibit tryptase in a bivalent manner, we chose the same sequence of amino
acids by analogy to RA-1002. The connective bridges of the L-leucyl-L-proline
dipeptides in RA-1003 and RA-1004 were xylyl and phenyl moieties.
Building block A – the precursor of RA-1003 – was synthesised via two routes
(routes I and II, Scheme 1). The N,N0-dicyclohexylcarbodiimide (DCC)-promoted
reaction of 1,4-bis(carboxymethyl)benzene (1) with L-leucine methyl ester hydro-
chloride (2) represents the first step in the successive synthesis depicted in route I.
After hydrolysis of 3 in a mixture of aqueous LiOH (1 M) and 1,2-dimethoxyethane
(DME) the resulting acid 4 was condensed with L-proline methyl ester hydro-
chloride (5, DIC-catalysis) yielding dipeptide methyl ester 6. The syntheses of
dipeptide esters containing a C-terminal L-proline moiety and the following con-
densation with alcohols or amines using carbodiimides as coupling reagents
strongly depend on the choice of appropriate reaction conditions [15]. In our hands,
the combination of N,N0-diisopropylcarbodiimide (DIC)=dichloromethane was the
most successful variation in both the synthesis of the dipeptide as well as the
syntheses of the appropriate dipeptide esters or amides. The utilization of DCC
at these steps was not satisfying, neither in dichloromethane nor in DMF (using
HOBt as an additional activating reagent).
Finally, hydrolysis of 6 (aqueous LiOH=DME) afforded building block A in
high yields. Compound 1 was synthesised from ꢀ,ꢀ0-dichloro-p-xylene following

Putative Dibasic Inhibitors of Human Mast Cell Tryptase
1161
Scheme 1. i) DCC=L-Leu-OMeÁHCl (2)=DIPEA=CH2Cl2, 0ꢁC, 34%; ii) LiOH (1 M)=DME, rt, 74%;
iii) DIC=L-Pro-OMeÁHCl (5)=DIPEA=CH2Cl2, 0ꢁC, 32%; iv) LiOH (1 M)=DME, rt, 82%;
v) DCC=5=DIPEA=CH2Cl2, 0ꢁC, 61%; vi) a) TFA=CH2Cl2, 0ꢁC, b) 1=DIC=DIPEA=CH2Cl2,
0ꢁC, 64%
Kolbe’s nitrile protocol. Acidic hydrolysis of the 1,4-bis(cyanomethyl)benzene
furnished 1 in high yields. L-Leucine methyl ester hydrochloride (2) and L-proline
methyl ester hydrochloride (5) were prepared according to the thionyl chloride
method [16].
The synthesis of 6 via route II initially consists of the condensation of N-Boc-L-
leucine with L-proline methyl ester hydrochloride (5). Cleavage of the tert-butyloxy-
carbonyl group by means of TFA=dichloromethane and subsequent transformation
of the ammonium into the amino group with DIPEA generated the unprotected
dipeptide methylester 8, which was condensed with 1 to give 6 subsequent to DIC-
activation of 1.
Route II impresses with a noticeable higher over-all yield (39%) compared to
route I (8%) concerning the synthesis up to intermediate 6. On the other hand, in
the case of phenylene derivative B the stepwise connection of each single building
block is more successful (route III, Scheme 2) than the condensation of terephthalic
acid with L-leucyl-L-proline methyl ester (8) which failed completely (route IV).
Thus, commercially available terephthaloyl chloride was aminolised by L-leucine
methyl ester hydrochloride (2) using an excess of DIPEA. The resulting methyl
ester 9 was subjected to the analogous procedure that was utilized in route I.
Hydrolysis of bisester 9 and condensation of the resulting acid 10 with two

1162
G. Radau
Scheme 2. i) 2=DIPEA=CH2Cl2, rt, 77%; ii) LiOH (1 M)=DME, rt, 95%; iii) DIC=5=DIPEA=
CH2Cl2, 0ꢁC, 58%; iv) LiOH (1 M)=DME, rt, 51%
equivalents of L-proline methyl ester hydrochloride (5) led to dipeptide deriva-
tive 11 in better yields compared to the synthesis of 6. Building block B was
obtained by hydrolysis of 11 – with an over-all yield of 22%.
In conclusion, we have examined the syntheses and the chemical properties of
precursors of potential bivalent inhibitors of human mast cell tryptase. Interestingly,
building blocks A and B display exact opposite behaviour during their syntheses
bearing only one subtle distinction in their chemical structure, namely the benzylic
methylene group. In the synthesis of building block A the synthetic pathway via the
central dipeptide 7 (route II) is to be favoured, whereas this procedure fails in
the preparation of building block B (route IV). The best way to synthesise B is the
stepwise manner (route III), which was less successful in the synthesis of A (route I).
Choosing the best of several practicable pathways, the peptide chemist profits
from his experience and synthetic talent, but it still stays nearly impossible to predict
the most successful route. Thus, each route has to be checked concerning parameters
like practicability, time-consumption, costs, and numbers of synthesis steps.
Experimental
General
Melting points are not corrected, Mikroheiztisch PHMK 80-2747 F. K€ustner Nachf. KG Dresden. IR
spectra (KBr): IR-Spektrometer Perkin-Elmer 1600 series FTIR. NMR spectra: Bruker DPX 300

Putative Dibasic Inhibitors of Human Mast Cell Tryptase
1163
(300 MHz), ꢁ (ppm), solvents: CDCl3, DMSO-d6, internal standard: TMS. Elemental analysis: Perkin-
Elmer Elemental Analyzer 2400 CHN, all compounds gave satisfactory elemental analyses.
Chromatography: cc: Merck silica gel 60 (0.063–0.200 mm); tlc: Merck aluminium foils silica gel
60 F254. Optical rotation: Polartronic D (Schmidt Haensch GmbH), determined with Na-D-line
(589.3 nm) at 23ꢁC. Abbreviations of amino acids follow the recommendations of the IUPAC-IUB
Joint Commission on Biochemical Nomenclature [17]. Other abbreviations: Boc: tert-butyloxycar-
bonyl, DCC: N,N0-dicyclohexylcarbodiimide, DCU: N,N0-dicyclohexylurea, DIC: N,N0-diisopropylcar-
bodiimide, DIPEA: diisopropylethylamine, DME: 1,2-dimethoxyethane, EtOAc: ethyl acetate, PE:
petroleum ether, TFA: trifluoroacetic acid.
1,4-Bis(carboxymethyl)benzene (1)
ꢀ,ꢀ0-Dichloro-p-xylene (15.00g, 85.7mmol) and 10.50 g of powdered sodium cyanide (214 mmol)
were stirred in 60 cm3 of triethylene glycol at 90ꢁC for 1 h. After cooling to room temperature, the
mixture was poured into 150 cm3 of H2O and extracted with 3Â75cm3 CH2Cl2. The combined CH2Cl2
layers were dried (Na2SO4), the solvent was evaporated, and the residue was recrystallised from
EtOAc=PE. Yield 11.6g (87%) of 1,4-bis(cyanomethyl)benzene, colourless crystals, mp 99ꢁC
1
(EtOAc=PE) (Ref. [18] 95–97ꢁC); H NMR (CDCl3): ꢁ ¼ 7.36 (4H, s, Harom), 3.77 (4H, s, CH2)
ppm; IR: 3035, 2936, 2916, 2248, 1518, 1424, 1414, 1126, 1021, 931, 782, 751, 475 cmÀ 1
.
1,4-Bis(cyanomethyl)benzene (5.50 g, 35.3mmol) was stirred in a mixture of 35 cm3 of acetic acid
and 175 cm3 of conc. HCl at 100ꢁC for 2 h. After cooling to 0ꢁC, the precipitate was collected, washed
with H2O, and recrystallised from H2O. Yield 6.1g (90%), colourless crystals, mp 252–253ꢁC
1
(EtOH=H2O) (Ref. [19] 253–254ꢁC). H NMR (DMSO-d6): ꢁ ¼ 12.31 (2H, s, COOH), 7.19 (4H, s,
Harom), 3.52 (4H, s, CH2) ppm; IR: 2800–3200, 2733, 2657, 1697, 1518, 1408, 1340, 1238, 1202,
1180, 899, 857, 784, 676, 515, 435 cmÀ 1
.
1,4-Phenylenedi(acetyl-L-leucine methyl ester) (3, C24H36N2O6)
To an ice-cooled mixture of 3.00 g of 1 (15.5 mmol) in 200 cm3 of CH2Cl2 a solution of 6.37 g of DCC
(30.9mmol) in 100 cm3 of CH2Cl2 was added dropwise over a period of 90min. After further 90min
of stirring, a mixture of 6.17 g of L-Leu-OMeÁHCl (2, 34.0 mmol) and 4.83 g of DIPEA (37.4mmol) in
100 cm3 of CH2Cl2 was added at 0ꢁC during 90 min. Stirring over night under warming up to room
temperature was followed by evaporation of the solvent. The residue was purified by column chro-
1
matography (EtOAc:PE¼ 5:1). Yield 2.33 g (34%), colourless crystals, mp 147–148.5ꢁC; H NMR
(CDCl3): ꢁ ¼ 7.27 (4H, s, Harom), 5.80 (2H, d, 8.2 Hz, NH), 4.59–4.66 (2H, m, ꢀ-H), 3.70 (6H, s,
OCH3), 3.59 (4H, s, Ar-CH2), 1.43–1.62 (6H, m, CH(CH3)2 þ ꢂ-H), 0.90 (6H, d, J ¼ 6.3 Hz,
CH(CH3)2), 0.89 (6H, d, J ¼ 6.3 Hz, CH(CH3)2) ppm; IR: 3261, 3069, 2956, 2870, 1745, 1641,
1553, 1439, 1353, 1276, 1248, 1203, 1165, 698, 541 cmÀ 1; [ꢀ]D ¼ À35.33 deg cm2 gÀ 1 (c ¼ 1%,
MeOH); M ¼ 448.56.
1,4-Phenylenedi(acetyl-L-leucine) (4, C22H32N2O6)
Compound 3 (2.24 g, 4.98mmol) was stirred in a mixture of 10cm3 of 1 M aqueous LiOH and 20cm3
of DME at room temperature for 2 h. The mixture was acidified (pH¼ 4) by addition of aqueous citric
acid (10%) and extracted with 2Â20cm3 of EtOAc. The combined organic layers were dried (Na2SO4)
and the filtrate was evaporated. The residue was purified by column chromatography
(CH2Cl2:EtOAc:PE:MeOH¼ 10:10:10:1! CH2Cl2:EtOAc:MeOH¼ 10:10:5). Yield 1.54g (74%), col-
ourless crystals, mp 184ꢁC; 1H NMR (DMSO-d6): ꢁ ¼ 12.48 (2H, s, COOH), 8.31 (2H, d, J ¼ 8.0 Hz,
NH), 7.15 (4H, s, Harom), 4.19–4.24 (2H, m, ꢀ-H), 3.42 (4H, d, J ¼ 14.1 Hz, Ar-CH2), 1.48–1.75 (6H,
m, CH(CH3)2 þ ꢂ-H), 0.88 (6H, d, J ¼ 6.4 Hz, CH(CH3)2), 0.81 (6H, d, J ¼ 6.4 Hz, CH(CH3)2) ppm;

1164
G. Radau
IR: 3343, 2800–3500, 2857, 2929, 2870, 1712, 1625, 1549, 1512, 1328, 1291, 1270, 1244, 1209, 1165,
1128, 788, 658, 603, 551, 503 cmÀ 1. [ꢀ]D ¼ À28.5deg cm2 gÀ 1 (c ¼ 2%, MeOH); M ¼ 420.51.
1,4-Phenylenedi(acetyl-L-leucyl-L-proline methyl ester) (6, C34H50N4O8)
Method A: Compound 4 (1.39 g, 3.31 mmol) was dissolved in 40 cm3 of CH2Cl2 and cooled to 0ꢁC. A
solution of 0.84g of DIC (6.62 mmol) in 10cm3 of CH2Cl2 was added dropwise over a period of
15min. The mixture was stirred at rt for 45 min (solution A) while in a second vessel 1.04 g of DIPEA
(8.01 mmol) in 10 cm3 of CH2Cl2 was added to 1.21 g of L-proline methyl ester hydrochloride (5,
7.28mmol) in 40cm3 of CH2Cl2 (solution B). The latter solution was added dropwise into solution A
at 0ꢁC (15 min) and this mixture was stirred for 16 h at rt and then it was evaporated in vacuo. The
resulting residue was purified by column chromatography to yield 0.68g (32%).
Method B: To an ice-cooled solution of 0.71 g of 1 (3.66 mmol) in 50 cm3 of CH2Cl2 0.92 g of DIC
(7.31 mmol) in 20 cm3 of CH2Cl2 were added dropwise (15 min). The mixture was stirred for 45min
and the dipeptide solution (L-Leu-L-Pro-OMe) was added dropwise at 0ꢁC (45 min). After stirring for
16h the mixture was evaporated in vacuo and the residue was purified by column chromatography
(EtOAc:PE ¼ 1:1 ! CH2Cl2:EtOAc:PE:MeOH¼ 10:10:10:1).
The dipeptide solution was obtained by treating 2.50 g of Boc-L-Leu-L-Pro-OMe (7.31 mmol) in a
TFA=CH2Cl2-mixture (30=80cm3) at rt for 3.5 h. The mixture was evaporated in vacuo and the residue
was dissolved in 20cm3 of methanol and the solvent was evaporated in vacuo again. To the remaining
oil a solution of 1.04 g of DIPEA (8.04 mmol) in 50 cm3 of CH2Cl2 was added and the mixture was
stirred for 15min at rt.
Yield 2.03 g (86%), colourless crystals, mp 106ꢁC; 1H NMR (CDCl3): ꢁ ¼ 7.19 (4H, s, Harom), 6.46
(2H, d, J ¼ 8.7 Hz, NH), 4.77–4.85 (2H, m, Leu-ꢀ-H), 4.46–4.51 (2H, m, Pro-ꢀ-H), 3.77–3.85 (2H, m,
Pro-N-CH2), 3.70 (6H, s, OCH3), 3.56–3.65 (2H, m, Pro-N-CH2), 3.50 (4H, s, Ar-CH2), 1.90–2.25
(8H, m, Pro-ꢂ-CH2 þ Pro-ꢃ-CH2) 1.58–1.70 (2H, sept, 6.5 Hz, CH(CH3)2), 1.46–1.52 (4H, m, Leu-ꢂ-
CH2), 0.95 (6H, d, J ¼ 6.5 Hz, CH(CH3)2), 0.90 (6H, d, J ¼ 6.5 Hz, CH(CH3)2) ppm; IR: 3289, 3064,
2956, 2872, 1747, 1631, 1543, 1514, 1448, 1366, 1320, 1276, 1197, 1175, 1097, 1024, 997, 724, 682,
597, 561 cmÀ 1; [ꢀ]D ¼ À103.16 deg cm2 gÀ 1 (c ¼ 1.5%, MeOH); M ¼ 642.78.
1,4-Phenylenedi(acetyl-L-leucyl-L-proline) (A, C32H46N4O8)
Compound 6 (2.42 g, 3.76mmol) was stirred in a mixture of 10 cm3 of 1 M aqueous LiOH and 20cm3 of
DME at room temperature for 2 h. The mixture was acidified (pH¼ 4) by addition of aqueous citric acid
(10%) and extracted with 2Â20 cm3 of EtOAc. The combined organic layers were dried (Na2SO4)
and the filtrate was evaporated. The residue was purified by column chromatography (CH2Cl2:
EtOAc:PE:MeOH ¼ 10:10:10:1! CH2Cl2:EtOAc:MeOH¼ 10:10:5). Yield 0.88 g (38%), colourless
crystals, mp 190ꢁC; 1H NMR (DMSO-d6): ꢁ ¼ 12.41 (2H, s br, COOH), 8.31 (2H, d, J ¼ 8.1 Hz, NH),
7.12 (4H, s, Harom), 4.49–4.56 (2H, m, Leu-ꢀ-H), 4.20–4.25 (2H, m, Pro-ꢀ-H), 3.60–3.71 (2H, m, Pro-
N-CH2), 3.40–3.50 (2H, m, Pro-N-CH2), 3.39 (4H, m, Ar-CH2), 2.06–2.18 (2H, m, Pro-ꢂ-CH2), 1.75–
1.95 (6H, m, Pro-ꢂ-CH2-ꢃ-CH2), 1.57–1.68 (2H, m, CH(CH3)2), 1.39–1.49 (4H, m, Leu-ꢂ-CH2), 0.89
(6H, d, J ¼ 6.6 Hz, CH(CH3)2), 0.84 (6H, d, J ¼ 6.6 Hz, CH(CH3)2) ppm; IR: 2850–3600, 3453, 2957,
1732, 1629, 1548, 1514, 1322, 1222, 1194, 668, 599, 562 cmÀ 1; [ꢀ]D ¼ À105.0deg cm2 gÀ 1 (c ¼ 1%,
MeOH); M ¼ 614.73.
Terephthaloyldi(L-leucine methyl ester) (9, C22H32N2O6)
To a suspension of 5.08g of terephthaloyl chloride (25.00 mmol) in 75 cm3 of CH2Cl2 a solution of
9.08g of L-leucine methyl ester hydrochloride (50.00 mmol) and 14.19g of DIPEA (110.00mmol) in
150 cm3 of CH2Cl2 was added at rt (40 min). After stirring for 16 h the mixture was extracted with
100 cm3 of 2 N HCl, the organic layer was dried (Na2SO4), and the solvent was evaporated in vacuo.

Putative Dibasic Inhibitors of Human Mast Cell Tryptase
1165
The resulting residue was recrystallised from EtOAc=EtOH. Yield 8.10g (77%), colourless crystals,
1
mp 234–235ꢁC; H NMR (CDCl3): ꢁ ¼ 7.76 (4H, s, Harom), 7.19 (2H, d, J ¼ 8.3 Hz, NH), 4.84–4.92
(2H, m, ꢀ-H), 3.80 (6H, s, OCH3), 1.65–1.80 (6H, m, CH(CH3)2 þ ꢂ-H), 0.97–1.00 (12H, 2d,
CH(CH3)2) ppm; IR: 3325, 2955, 2870, 1745, 1634, 1549, 1502, 1340, 1278, 1210, 1162, 1017,
872, 846, 748, 730, 640 cmÀ 1; [ꢀ]D ¼ À18.66 (c ¼ 2%, MeOH); M ¼ 420.51.
Terephthaloyldi(L-leucine) (10, C20H28N2O6)
Compound 9 (1.57 g, 3.74mmol) was stirred in a mixture of 10cm3 of 1 M aqueous LiOH and 20cm3
of DME at room temperature for 2 h. The mixture was acidified (pH¼ 4) by addition of aqueous citric
acid (10%) and extracted with 2Â20 cm3 of EtOAc. The combined organic layers were dried (Na2SO4)
and the filtrate was evaporated. The residue was purified by column chromatography (CH2Cl2:
EtOAc:PE:MeOH ¼ 10:10:10:1! CH2Cl2:EtOAc:MeOH¼ 10:10:5). Yield 1.38 g (95%), colourless
crystals, mp 240–242ꢁC; 1H NMR (DMSO-d6): ꢁ ¼ 12.41 (2H, s br, COOH), 8.72 (2H, d, J ¼ 7.9 Hz,
NH), 7.96 (4H, s, Harom), 4.42–4.48 (2H, m, ꢀ-H), 1.59–1.82 (6H, m, CH(CH3)2 þ ꢂ-H), 0.93 (6H, d,
J ¼ 6.2 Hz, CH(CH3)2), 0.89 (6H, d, J ¼ 6.2 Hz, CH(CH3)2) ppm; IR: 3382, 2961, 2872, 2400–3500,
1728, 1632, 1542, 1500, 1270, 1234, 1170, 1017, 968, 870, 847, 748, 713, 660, 598 cmÀ 1
[ꢀ]D ¼ À5.87deg cm2 gÀ 1 (c ¼ 2%, MeOH); M ¼ 392.46.
;
Terephthaloyldi(L-leucyl-L-proline methyl ester) (11, C32H46N4O8)
Compound 10 (3.44 g, 8.79mmol) was dissolved in 130cm3 of CH2Cl2 and cooled to 0ꢁC. A solution
of 2.22g of DIC (17.58 mmol) in 30cm3 of CH2Cl2 was added dropwise over a period of 30min. The
mixture was stirred at rt for 1 h (solution A) while in a second vessel 2.74g of DIPEA (21.27 mmol) in
30cm3 of CH2Cl2 were added to 3.20 g of L-proline methyl ester hydrochloride (5, 19.34mmol) in
100 cm3 of CH2Cl2 (solution B). The latter solution was added dropwise into solution A at 0ꢁC
(45 min) and this mixture was stirred for 16h at rt and then the solvent was evaporated in vacuo.
The resulting residue was purified by column chromatography (EtOAc:PE¼ 1:1 ! EtOAc:PE ¼ 2:1).
1
Yield 3.13 g (58%), colourless crystals, mp 195–203ꢁC; H NMR (CDCl3): ꢁ ¼ 7.69–7.84 (4H, m,
Harom), 7.03 (2H, d, NH), 5.01–5.20 (2H, m, Leu-ꢀ-H), 4.41–4.56 (2H, m, Pro-ꢀ-H), 3.85–4.00 (2H,
m, Pro-N-CH2), 3.69 (6H, s, OCH3), 3.55–3.66 (2H, m, Pro-N-CH2), 1.90–2.25 (8H, m, Pro-ꢂ-
CH2 þ Pro-ꢃ-CH2), 1.40–1.85 (6H, m, Leu-ꢂ-CH2 þ CH(CH3)2), 0.89–1.07 (12H, m, CH(CH3)2)
ppm; IR: 3454 (NH), 2956, 2872, 1745 (C¼O), 1632 (C¼O), 1544, 1498, 1439, 1290, 1366, 1343,
1198, 1174, 872, 729, 606 cmÀ 1; [ꢀ]D ¼ À59.41 deg cm2 gÀ 1 (c ¼ 2%, MeOH); M ¼ 614.73.
Terephthaloyldi(L-leucyl-L-proline) (B, C30H42N4O8)
Compound 11 (2.08 g, 3.39 mmol) was stirred in a mixture of 10cm3 of 1 M aqueous LiOH and 20cm3
of DME at room temperature for 2 h. The mixture was acidified (pH¼ 4) by addition of aqueous
citric acid (10%) and extracted with 2Â20cm3 of EtOAc. The combined organic layers were dried
(Na2SO4) and the filtrate was evaporated. The residue was purified by column chromatography
(CH2Cl2:EtOAc:PE:MeOH¼ 10:10:10:1! CH2Cl2:EtOAc:MeOH¼ 10:10:5). Yield 1.01g (51%), col-
1
ourless crystals, mp 247ꢁC (dec); H NMR (DMSO-d6): ꢁ ¼ 12.48 (2H, s br, COOH), 8.71 (2H, d,
J ¼ 7.8 Hz, NH), 7.97 (4H, s, Harom), 4.72–4.81 (2H, m, Leu-ꢀ-H), 4.18–4.30 (2H, m, Pro-ꢀ-H),
3.73–3.85 (2H, m, Pro-N-CH2), 3.56–3.62 (2H, m, Pro-N-CH2), 2.10–2.20 (2H, m, Pro-ꢂ-CH2),
1.65–2.00 (10H, m, Pro-ꢂ-CH2-ꢃ-CH2 þ Leu-ꢂ-CH2), 1.45–1.53 (2H, m, CH(CH3)2), 0.90 (6H, d,
J ¼ 6.6 Hz, CH(CH3)2), 0.86 (6H, d, J ¼ 6.6 Hz, CH(CH3)2) ppm; IR: 3445, 2958, 1729, 1633, 1541,
1497, 1451, 1317, 1224, 1193, 870, 668, 600 cmÀ 1; [ꢀ]D ¼ À42.16 deg cm2 gÀ 1 (c ¼ 2%, MeOH);
M ¼ 586.67.

1166
G. Radau: Putative Dibasic Inhibitors of Human Mast Cell Tryptase
Acknowledgement
The author would like to thank the Fonds der Chemischen Industrie and the BMBF for financial
support.
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