Synthesis and biochemical investigation of scyphostatin analogues as inhibitors of neutral sphingomyelinase

Article abstract of DOI:10.1016/S0968-0896(01)00165-1

The sphingolipid ceramide is considered to be an important intracellular mediator. However, many aspects of its action and the role of several different ceramide generating sphingomyelinases are still unclear. Recently, we reported on the synthesis of the first selective irreversible inhibitor of the neutral sphingomyelinase (N-SMase), as well as the identification of Manumycin A and some of its analogues as irreversible inhibitors of N-SMase. For the development of pharmacologically interesting competitive inhibitors of N-SMase, structure-activity studies are essential. Herein we show the synthesis and enzymatic investigation of two scyphostatin analogues 3a and 3b, revealing the importance of the primary hydroxy group in compound 2 for N-SMase inhibition.

Full text of DOI:10.1016/S0968-0896(01)00165-1

Bioorganic & Medicinal Chemistry9 (2001) 2901–2904
Synthesis and Biochemical Investigation of Scyphostatin
Analogues as Inhibitors of Neutral Sphingomyelinase
Christoph Arenz, Michael Gartner, Veit Wascholowski and Athanassios Giannis*
Institut fur Organische Chemie, Universitat Karlsruhe, Richard-Willstatter Allee 2, 76128Karlsruhe, Germany
¨
¨
¨
Received 21 March 2001; accepted 8 May2001
Abstract—The sphingolipid ceramide is considered to be an important intracellular mediator. However, manyaspects of its action
and the role of several different ceramide generating sphingomyelinases are still unclear. Recently, we reported on the synthesis of
the first selective irreversible inhibitor of the neutral sphingomyelinase (N-SMase), as well as the identification of Manumycin A
and some of its analogues as irreversible inhibitors of N-SMase. For the development of pharmacologicallyinteresting competitive
inhibitors of N-SMase, structure–activity studies are essential. Herein we show the synthesis and enzymatic investigation of two
scyphostatin analogues 3a and 3b, revealing the importance of the primaryhydroxygroup in compound 2 for N-SMase inhibition.
# 2001 Elsevier Science Ltd. All rights reserved.
Introduction
ceramide. Recently, we reported on the synthesis of the
spiroepoxide 2,11,12 which was synthesized as an analo-
gue of the natural product scyphostatin 1.13ꢀ15 The lat-
ter is known as a potent inhibitor of the membrane-
bound neutral sphingomyelinase (N-SMase). In fact,
analogue 2 inhibits N-SMase, but contraryto scyphos-
tatin, it is an irreversible inhibitor of this enzyme.
Moreover, we could identifythe antibiotic Manumycin
A and two of its analogues as potent irreversible N-
SMase inhibitors16 (Fig. 1).
The sphingolipid ceramide is a putative second messen-
ger and can be generated from sphingomyelin—an
important constituent of eucaryotic membranes-
through the action of various sphingomyelinases.1,2
These enzymes can be activated by various cytokines, or
byradiation, heat and oxidative stress leading to an
increased ceramide production. It is suggested, that cer-
amide acts as a second messenger and triggers or mod-
ulates
a number of fundamental processes like
programmed cell death (apoptosis), the cell cycle or
inflammatoryprocesses in various tissues and cell lines. 3
Moreover, recent studies stronglysuggest a vital role for
4,5
ceramide as a keymediator in tumor suppression.
However, numerous aspects of ceramide mediated sig-
nal transduction, especiallythose related to apoptosis
remain unclear.6,7 Furthermore the question, whether
the plasma membrane-bound Mg2+ dependent neutral
sphingomyelinase (N-SMase), out of several described
neutral sphingomyelinases8ꢀ10 or the lysosomal acid
sphingomyelinase (A-SMase) is most important for sti-
mulus-induced ceramide production is discussed con-
troversially.1,6 Selective inhibitors of the different
sphingomyelinases would be valuable tools for the
investigation of the biological role of these enzymes and
*Corresponding author. Tel.: +49-721-608-3209; fax: +49-721-608-
7652; e-mail: giannis@ochhades.chemie.uni-karlsruhe.de
Figure 1.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00165-1

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C. Arenz et al. / Bioorg. Med. Chem. 9 (2001) 2901–2904
Regarding the varietyof biological effects possibly
triggered byceramide, N-SMase emerges as an interest-
ing target for the development of anti-inflammatory
drugs.13 Therefore, the synthesis of competitive inhibi-
tors, which in comparison to irreversible inhibitors are
more likelyto fulfill the requirements of a ‘drug’, is well
justified.
derivative 6, which was obtained byLiAlH -reduction
4
of aminosalicylic acid methyl ester. Treatment of the
resulting compounds 7a,b with sodium periodate in a
mixture of methanol/water and dichloromethane affor-
ded the spiroepoxides 3a and 3b as mixtures of the
corresponding diastereomeres.
Compounds 3a and 3b were tested upon N-SMase and
A-SMase inhibition, as described,11,16 using a raw pre-
paration of rat brain microsomes. To determine the
inhibitorypotencytowards N-SMase theywere tested
with and without pre-incubation for 90 min with the
enzyme. The latter likely represents the ability of the
inhibitor to compete with the substrate for binding at
the active site of the enzyme, whereas the assay with
pre-incubation of the inhibitor with the enzyme in the
absence of substrate represents the kinetics of the irre-
versible inhibition. In fact both derivatives 3a,b proved
to be weak irreversible N-SMase inhibitors (Table 1).
Compared to the epoxide 2, which was derived from d-
serine, these results clearlyshow the importance of the
primaryhydroxygroup. Furthermore, no inhibition of
A-SMase was observed (data not shown).
Our first aim is to gain more insights into the mode of
N-SMase inhibition bythe different inhibitors and to
clarifythe role of the different functional groups which
are involved. Our investigations with analogues of
Manumycin A revealed, that inhibitory potency of such
compounds is stronglyinfluenced bythe nature of the
hydrophobic side chain.16
In the present paper, we synthesized two analogues of 2
in which the primaryhydroxygroup is replaced bya
hydrogen or a phenyl function (Scheme 1). Furthermore,
the abilityof both compounds to inhibit N-SMase was
investigated.
Results and Discussion
The amino acids d-alanine 4a and d-phenylalanine 4b
were acylated with decanoyl chloride under Schotten–
Baumann conditions to give N-decanoyl-d-alanine 5a
and N-decanoyl-d-phenylalanine 5b. Subsequently, the
acylated amino acids 5 a,b were coupled with the aniline
Conclusion
The inhibitorypotencyof the epoxides
3a and 3b
towards N-SMase is drasticallyreduced, compared to
the known inhibitor 2, indicating that the primary
hydroxy group is crucial for a enzyme inhibition.
These insights and findings will greatlycontribute in the
development of potent competitive inhibitors of N-
SMase and mayfinallyprovide a means for the devel-
opment of novel antiinflammatorytherapeutic strate-
gies. Now, we are concentrating our research interests
on the synthesis of selective competitive inhibitors of N-
SMase, which in our knowledge are not identified so far.
Experimental
General remarks
Melting points were determined in open capillaries
1
using a Buchi 535 apparatus and are uncorrected. H
and 13C NMR spectra were recorded on a Bruker AC
250, AM 400, or DRX 500 spectrometer at room tem-
perature. Mass spectra and high-resolution mass spectra
(HR-MS) were measured on a Finnigan MAT MS70
spectrometer. Materials: Solvents were dried bystan-
dard methods and stored over molecular sieves. For
column chromatographysilica gel (40 ꢁ60 mm, Merck
AG) was used. Commercial reagents were used without
further purification.
Scheme 1. Synthesis of the scyphostatin analogues 3a,b: (a) H2O,
THF, Na2CO3, 18 h, rt, 58%/63%; (b) DCC, HOBt, DMF, 18 h, 26%/
39%; (c) NaIO4, MeOH/H2O, 3 h, rt, 26%/21%.
Table 1. Inhibition of N-SMase bydifferent spiroepoxides 2, 3a and
3b (200 mM in pre-incubation buffer, 100 mM final concentration)
Inhibition without
pre-incubation (%)
Inhibition with
pre-incubation (%)
N-Decanoyl-D-alanine (5a). To a suspension of d-ala-
nine (5.00 g, 56.1 mmol) and sodium bicarbonate (8.93 g,
84.2 mmol) in water (100 mL) and tetrahydrofuran
(50 mL) decanoyl chloride (12.62 mL, 11.77 g,
61.7 mmol) was slowlyadded. After stirring for 18 h
at room temperature tetrahydrofuran was carefully
2
3a
3b
17
12
5
88
33
23
The pre-incubation time was 90 min.

C. Arenz et al. / Bioorg. Med. Chem. 9 (2001) 2901–2904
2903
removed under reduced pressure. The resulting solution
was acidified to a pH of 1–2 with 6 N hydrochloric acid
until a white solid was precipitated. After filtration and
drying the crude product was recrystallized from diethyl
ether. White solid 7.92 g (32.5 mmol, 58%). Mp 89 ꢂC,
Rf=0.70 (dichloromethane/methanol 10:1). 1H NMR
(CDCl3, 500 MHz): d 0.88 (t, 3H, 3J=7.0 Hz, CH3),
temperature and stirred for additional 18 h. The pre-
cipitate was filtered off and washed twice with ice-cold
THF. Then the solvent was removed under reduced
pressure and the residue was chromatographed (silica
gel 40–60, dichloromethane/methanol 12:1). Chromato-
graphyyielded a white solid (1.77 g, 4.9 mmol, 26%).
Mp 178 ꢂC, Rf=0.25 (dichloromethane/methanol 12:1).
3
1.26–1.33 (m, 12H, 6ꢃCH2), 1.46 (d, 3H, J=7.1 Hz,
1H NMR DMSO-d6, 500 MHz):
d 0.84 (t, 3H,
3
CH3), 1.60–1.66 (m, 2H, CH2), 2.24 (t, 2H, J=7.7 Hz,
3J=6.8 Hz, CH3), 1.22–1.27 (m, 15H, 6ꢃCH2 and
1ꢃCH3), 1.45–1.48 (m, 2H, CH2), 2.10 (t, 2H,
3J=7.4 Hz, CH2C¼O), 4.37 (m, 1H, CHa), 4.43 (d, 2H,
3J=5.4 Hz, CH2OH), 4.96 (t, 1H, 3J=5.4 Hz, CH2OH);
CH2C¼O), 4.55–4.61 (m, 1H, CHa), 6.16 (m, 1H, NH),
9.37 (br, 1H, COOH) ppm. 13C NMR (CDCl3,
125.8 MHz): d 14.09 (CH3), 18.01 (CH3), 22.66 (CH2),
25.55 (CH2), 29.18 (CH2), 29.25 (CH2), 29.30 (CH2),
29.42 (CH2), 31.85 (CH2), 36.45 (CH2), 48.29 (CH),
174.04 (C), 176.01 (C) ppm, HR-MS (70 eV, 100 ꢂC):
calcd for C13H25NO3(M+): m/z 243.1834; found: m/z
243.1838.
3
6.65 (d, 1H, J=8.6 Hz, NH), 7.29 (dd, 1H, J=8.6 Hz
and J=2.4 Hz, CHarom.), 7.44 (d, 1H, J=2.0 Hz,
CHarom.), 7.98 (d, 1H, J=7.4 Hz, CHarom.), 9.10 (s, 1H,
NH), 9.64 (s, 1H, OH) ppm. 13C NMR (125.8 MHz,
(DMSO-d6): d 13.94 (CH3), 18.29 (CH3), 22.09 (CH2),
25.20 (CH2), 28.65 (CH2), 28.75 (CH2), 28.81 (CH2),
28.90 (CH2), 31.27 (CH2), 35.05 (CH2), 48.69 (CH),
58.11 (CH2), 114.20 (CH), 118.72 (CH), 119.11 (CH),
128.66 (C), 130.59 (C), 149.98 (C), 170.64 (C), 172.04
(C) ppm. HR-MS (70 eV, 180 ꢂC): calcd for
C20H32N2O4(M+): m/z 364.2362; found: m/z 364.2356.
N-Decanoyl-D-phenylalanine (5b). Preparation was
taken out analogouslyas described for ( 5a). White solid
(63% yield). Mp 118 ꢂC, Rf=0.70 (dichloromethane/
1
methanol 12:1). H NMR (CD3OD, 500 MHz): d 0.80
3
(t, 3H, J=7.0 Hz, CH3), 1.16–1.23 (m, 12H, 6ꢃCH2),
1.42–1.47 (m, 2H, CH2), 2.06 (t, 2H, 3J=7.6 Hz,
CH2C¼O), 2.95 (dd, 1H, 2J=13.9 Hz, 3J=7.1 Hz,
CHHPh), 3.12 (dd, 1H, 2J=13.9 Hz, 3J=5.3 Hz,
CHHPh), 4.57–4.60 (m, 1H, CHa), 7.09–7.20 (m, 5H,
CHarom.) ppm. 13C NMR (CD3OD, 125.8 MHz): d 13.91
(CH3), 22.52 (CH2), 25.51 (CH2), 29.05 (CH2), 29.15
(CH2), 29.21 (CH2), 29.30 (CH2), 31.74 (CH2), 36.28
(CH2), 37.39 (CH2), 53.97 (CH), 126.65 (CH), 128.24
(2ꢃCH), 129.19 (2ꢃCH), 136.69 (C), 173.85 (C), 175.23
(C) ppm. HR-MS (70 eV, 140 ꢂC): calcd for
C19H29NO3(M+): m/z 319.2147; found: m/z 319.2137.
Decanoic acid [2-phenyl-1-(R)-(4-hydroxy-3-hydroxymethyl-
phenylcarbamoyl)-ethyl]-amide (7b). Preparation was
taken out analogouslyas described for ( 7a). White solid
(39% yield). Mp 116 ꢂC, Rf=0.40 (dichloromethane/
methanol 10:1). 1H NMR (DMSO-d6, 500 MHz): d 0.84
(t, 3H, 3J=7.0 Hz, CH3), 1.08–1.09 (m, 2H, CH2), 1.16–
1.25 (m, 10H, 5ꢃCH2), 1.33–1.39 (m, 2H, CH2), 2.03 (t,
3
3
2H, J=7.3 Hz, CH2C¼O), 2.81 (dd, 1H, J=13.7 Hz,
3J=9.8 Hz, CHHPh), 3.00 (dd, 1H, 2J=13.7 Hz,
3J=4.9 Hz, CHHPh), 4.44 (d, 2H, 3J=4.1 Hz, CH2OH),
4.60–4.65 (m, 1H, CHa), 4.96–4.97 (m, 1H, CH2OH),
6.66 (d, 1H, J=8.6 Hz, CHarom.), 7.15–7.17 (m, 1H,
CHarom.), 7.22–7.31 (m, 5H, CHarom. (Phe)), 7.42–7.43
5-Amino-2-hydroxy-benzylalcohol (6). A solution of 5-
amino-2-hydroxy-benzoic acid methyl ester (10.03 g,
60.0 mmol) in absolute tetrahydrofuran (50 mL) was
added dropwise to an ice-cooled and well-stirred sus-
pension of lithium aluminium hydride (4.55 g,
120.0 mmol) in absolute tetrahydrofuran (150 mL).
Then the yellow reaction mixture was allowed to warm
up at room temperature and stirred for 3 h. After the
reaction was complete (TLC control) the mixture was
cooled again to 0 ꢂC and quenched with isopropanol.
The resulting suspension was filtered under nitrogen
and the solid residue was washed two times with dry
tetrahydrofuran. The solvent was removed in vacuo and
the benzyl alcohol 5 was obtained as a yellow air-sensi-
tive solid (7.71 g, 55.4 mmol, 92%), which was stored
under argon atmosphere. Rf=0.30 (dichloromethane/
3
(m, 1H, CHarom.), 8.07 (d, 1H, J=8.4 Hz, NH (Phe)),
9.11 (s, 1H, NH), 9.77 (s, 1H, OH) ppm. 13C NMR
(125.8 MHz, DMSO-d6): d 13.94 (CH3), 22.08 (CH2),
25.19 (CH2), 28.45 (CH2), 28.65 (CH2), 28.78 (CH2),
28.85 (CH2), 31.27 (CH2), 35.15 (CH2), 37.86 (CH2),
54.52 (CH), 58.10 (CH2), 114.21 (CH), 118.84 (CH),
119.20 (CH), 126.18 (CH), 127.94 (2ꢃCH), 128.69 (C),
129.16 (2ꢃCH), 130.44 (C), 137.94 (C), 150.08 (C),
169.57 (C), 172.14 (C) ppm. HR-MS (70 eV, 220 ꢂC):
calcd for C26H36N2O4(M+): m/z 440.2675; found: m/z
440.2651.
Decanoic acid [1-(R)-(8-oxo-1-oxaspiro[2,5]octa-4,6-
diene-5-ylcarbamoyl)-ethyl]-amide (3a). Compound 7a
(300 mg, 0.82 mmol) was dissolved in a mixture of di-
chloromethane/methanol 3:2 (30 mL) and treated with a
0.3 M aqueous solution of sodium periodate (6.8 mL,
2.05 mmol). After stirring for 3 h in the dark, the
organic layer was separated and the water phase was
extracted three times with dichloromethane. Then the
combined organic layers were dried over sodium sulfate
and the solvent was removed. The crude product was
chromatographed on silica gel (dichloromethane/
methanol 12:1) to afford the product as an inseparable
mixture of diastereomeres in the ratio of 59:41 (77 mg,
0.21 mmol, 26%). Rf=0.35 (dichloromethane/methanol
1
methanol 7:1). H NMR (CD3OD, 250 MHz): d 4.62 (s,
2H, CH2OH), 6.61–6.62 (m, 2H, CHarom.), 6.79–6.80
(m, 1H, CHarom.) ppm. HR-MS (70 eV, 110 ꢂC): calcd
for C7H9NO2(M+): m/z 139.0633; found: m/z 139.0629.
Decanoic acid [1-(R)-(4-hydroxy-3-hydroxymethylphenyl-
carbamoyl)-ethyl]-amide (7a). To an ice-cooled solution
of N-decanoyl-d-alanine 5a (4.50 g, 18.5 mmol), 5-
amino-2-hydroxy-benzylalkohol 2 (3.86 g, 27.8 mmol)
and HOBt (3.40 g, 22.2 mmol) in dryDMF (100 mL)
DCC (4.58 g, 22.2 mmol) was added. After stirring for
1 h at 0 ꢂC, the mixture was allowed to warm up at room

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C. Arenz et al. / Bioorg. Med. Chem. 9 (2001) 2901–2904
12:1). 1H NMR (CDCl3, 500 MHz): d 0.86 (t, 3H,
3J=7.0 Hz, CH3), 1.23–1.47 (m, 15H, 6ꢃCH2, 1ꢃCH3),
1.52–1.59 (m, 2H, CH2), 2.09–2.28 (m, 2H, CH2C¼O),
3.20–3.21 (m, 2H, CH2 epox.), 4.49–4.55 (m, 1H, CHa),
6.29–6.30 (m, 0.59H, CHring), 6.31–6.32 (m, 0.41H,
(CH2), 36.42 (CH2), 38.44 (CH2), 54.87 (CH), 59.45
(CH2), 79.83 (C), 127.05 (CH), 128.55 (2ꢃCH), 129.31
(2ꢃCH), 129.85 (CH), 136.16 (C), 138.23 (CH), 140.04
(C), 144.28 (CH), 171.38 (C), 174.04 (C), 185.17 (C)
ppm.
HR-MS
(70 eV,
200 ꢂC):
calcd
for
3
CHring), 6.49 (d, 0.59H, J=7.4 Hz, NH (Ala)), 6.54 (d,
C26H34N2O4(M+): m/z 438.2519; found: m/z 438.2548.
0.41H, 3J=7.4 Hz, NH, (Ala)), 6.70 (br, 0.59H, CHring),
6.83 (br, 0.41H, CHring), 7.04 (dd, 0.59H, J=10.2 Hz
and J=3.0 Hz, CHring), 7.10 (dd, 0.41H, J=10.2 Hz and
J=3.0 Hz, CHring), 7.85 (br, 0.59H, NH), 7.95 (br,
0.41H, NH) ppm. 13C NMR (CDCl3, 125.8 MHz, major
diastereomer): d 14.08 (CH3), 18.18 (CH3), 22.63 (CH2),
25.59 (CH2), 29.20 (CH2), 29.24 (CH2), 29.30 (CH2),
29.42 (CH2), 31.82 (CH2), 36.44 (CH2), 49.06 (CH),
59.95 (CH2), 79.90 (C), 130.24 (CH), 138.81 (CH),
139.88 (C), 144.58 (CH), 172.09 (C), 173.82 (C), 185.38
(C) ppm. Minor diastereomer: d 14.08 (CH3), 18.18
(CH3), 22.63 (CH2), 25.59 (CH2), 29.20 (CH2), 29.24
(CH2), 29.30 (CH2), 29.42 (CH2), 31.82 (CH2), 36.44
(CH2), 49.11 (CH), 59.53 (CH2), 79.87 (C), 129.99 (CH),
138.35 (CH), 140.11 (C), 144.47 (CH), 172.34 (C),
173.94 (C), 185.20 (C) ppm. HR-MS (70 eV, 160 ꢂC):
calcd for C20H30N2O4(M+): m/z 362.2206; found m/z
362.2213.
Acknowledgements
C.A. and V.W. are grateful for grants of the
Landesgraduierten Forderung, Baden-Wurttemberg.
References and Notes
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Decanoic acid [2-phenyl-1-(R)-(8-oxo-1-oxaspiro[2,5]octa-
4,6-diene-5-ylcarbamoyl)-ethyl]-amide (3b). Preparation
was taken out analogouslyas described for 3a. Product
was obtained as an inseparable mixture of correspond-
ing diastereomeres in the ratio of 63:37 (21% yield).
Rf=0.40 (dichloromethane/methanol 12:1). 1H NMR
(CDCl3, 500 MHz): d 0.85–0.88 (m, 3H, CH3), 1.04–1.36
(m, 12H, 6ꢃCH2), 1.39–1.58 (m, 2H, CH2), 2.01–2.25
(m, 2H, CH2), 2.93–3.08 (m, 2H, CH2–Ph), 3.11–3.14
(m, 2H, CH2epox.), 4.70–4.85 (m, 1H, CHa), 6.21–6.24
(m, 0.37H, CHring), 6.26–6.28 (m, 0.63H, CHring), 6.61
(br, 0.63H, CHring), 6.66 (br, 0.37H, CHring), 6.95–7.00
(m, 1H, CHring), 7.12–7.31 (m, 6H, CHarom. (Phe)), 7.58
(br, 0.63H, NH), 7.85 (br, 0.37H, NH) ppm. 13C NMR
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(CDCl3, 125.8 MHz, major-diastereomer):
d 14.10
(CH3), 22.65 (CH2), 25.56 (CH2), 29.15 (CH2), 29.27
(CH2), 29.34 (CH2), 29.43 (CH2), 31.85 (CH2), 36.42
(CH2), 38.37 (CH2), 54.55 (CH), 59.86 (CH2), 79.83 (C),
127.05 (CH), 128.55 (2ꢃCH), 129.34 (2ꢃCH), 130.12
(CH), 136.16 (C), 138.63 (CH), 139.85 (C), 144.53 (CH),
170.92 (C), 173.79 (C), 185.36 (C) ppm. Minor-diaster-
eomer: d 14.10 (CH3), 22.65 (CH2), 25.56 (CH2), 29.15
(CH2), 29.27 (CH2), 29.34 (CH2), 29.43 (CH2), 31.85