Muraymycins, novel peptidoglycan biosynthesis inhibitors: Synthesis and SAR of their analogues

Article abstract of DOI:10.1016/S0960-894X(03)00671-1

A series of Muraymycin analogues was synthesized. These analogues showed excellent antimicrobial activity against gram-positive organisms. These analogues also showed excellent inhibitory activity against the target peptidoglycan biosynthesis enzyme MraY,

Full text of DOI:10.1016/S0960-894X(03)00671-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3345–3350
Muraymycins, Novel Peptidoglycan Biosynthesis Inhibitors:
Synthesis and SAR of Their Analoguesy
Ayako Yamashita,* Emily Norton, Peter J. Petersen, Beth A. Rasmussen, Guy Singh,
{
Youjin Yang, Tarek S. Mansour andDouglas M. Ho
Chemical Sciences and Infectious Diseases, Wyeth Research, Pearl River, NY 10965, USA
Received24 December 2002; accepted8 May 2003
Abstract—A series of Muraymycin analogues was synthesized. These analogues showed excellent antimicrobial activity against
gram-positive organisms. These analogues also showedexcellent inhibitory activity against the target peptidoglycan biosynthesis
enzyme MraY, the cell membrane associatedtransglycosylase responsible for the formation of LipidII.
# 2003 Elsevier Ltd. All rights reserved.
Over the past years, a large number of naturally derived
semi-synthetic or synthetic antibiotics in various classes
such as glycopeptides, tetracyclines and macrolides,
have improvedthe quality of these compounds for the
treatment of infections causedby a broadrange of bac-
teria. However, despite the historical success of this
strategy in combating bacterial infections, relatively few
antibacterial agents of microbial origin are currently
being pursuedin the clinic. This is primarily attributable
to the considerable difficulty in identifying structurally
novel antibiotics with potent andselective antimicrobial
properties.1 On the other hand, as a consequence to the
indiscriminate use of antibiotics, the emergence of
multi-drug resistant pathogens has resulted in an epi-
demic situation. This problem, therefore, necessitated
the search for new antibacterial agents by employing
different strategies, such as relying on advanced screen-
ing technologies, understanding structural basis for the
design of new generation of antibacterial compounds,2
or unraveling the understanding of resistance mechan-
isms3 to current antibacterial agents in the hope that a
new generation of antimicrobial agents emerges.
This paper outlines our approach to new antibacterial
agents, basedon expanding the structure activity rela-
tionship of muraymycins4,5 (1a,b and 2a,b) via the
development of synthetic routes toward the total synth-
esis of muraymycins, a novel class of peptidoglycan
biosynthesis inhibitors,6 with potent activity against
gram-positive organisms with minimum inhibitory con-
centration (MIC) of 1–8 mg/mL. The new target analo-
gues differ from naturally occurring muraymycins by
replacement of the cyclic arginine amino acidwith argi-
0
nine andby the removal of the 5 -amino ribose sugar
moiety 3 as well as the removal of the lipophilic side
chain X (Fig. 1). During this process, we foundthat the
truncatedmuraymycins ( 19, Scheme 4) were as active as
the natural products against gram positive organisms.
A synthetic route was developed to allow for rapid
introduction of various amino acids and linkers in the
acyclic portion of muraymycins. The first amino acid
moiety (glycine) was planned to be introduced by aldol
reaction of an uridyl-50 aldehyde with a suitable glycine
derivative. Toward this end, aldehyde 47 was syn-
thesizedfrom d-uridine. Aldol reaction between 4 and
an anion of dibenzylglycine tert-butyl ester,8 generated
by treatment with LDA in THF at ꢀ78 ꢁC, afforded a
mixture of four diastereomers, which were separated by
chromatography (Scheme 1). The two major compo-
nents (5 and 6) were deprotected by catalytic hydro-
genation, to form their free amines (7 and 8). The
absolute stereochemistry of the newly createdchiral
centers at positions 5 and6 in 7 and 8 was deter-
minedby the X-ray crystallographic analyses of their
yA part of this work was presentedat the 41st Interscience Conference
on Antimicrobial Agents andChemotherapy, Chicago, IL, USA.
Yamashita, A.; Norton, E.; Labthavikul, P.; Petersen, P. J.; Rasmus-
sen, B. A.; Singh, G.; Yang, Y.; Mansour, T. S. Abstract No. 1161.
*Corresponding author. Fax: +1-845-602-5561; e-mail: yamashita@
wyeth.com
{Current address: Director, Small Molecule X-ray Facility, Depart-
ment of Chemistry, Princeton University, Princeton, NJ 08544, USA.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00671-1

3346
A. Yamashita et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3345–3350
Scheme 2. (a) H2N(CH2)2CH(OEt)2, HOBT, EDC, DIPA, THF;
(b) HCl, THF.
Figure 1.
Scheme 3. (a) Isobutylene, c-H2SO4, CH2Cl2; (b) piperidine, rt, 1 h;
(c) 15, triphosgene, DIPA, rt; (d) TFA, CH2Cl2, 0 ꢁC.
andR substituents of 11. Further progress of 18a was
2
achievedby removal of the CBZ group by catalytic
hydrogenation, to give the free amine 19a. Coupling
between 17 and 18a under the conditions using hydroxy-
benzotriazole (HOBT) and1-(3i-md ethylaminopro-
pyl)-3-ethylcarbodiimide (EDC) allowed for the
construction of the fully protectedfinal target molecule
20a in goodisolatedyield( Scheme 4).11 In a similar
fashion, 20b and 20c were isolated. The (5S,6S) diastereo-
mer 8 was also successfully convertedto the corre-
sponding threonine intermediate 21a, then to the
unprotectedamine 22a. This result suggests that the
stereochemical difference between 7 and 8 at the C5
position has no outcome in influencing the reductive
amination and deprotection steps under the reported
experimental conditions.
Scheme 1. (a) (PhCH2)2NCH2CO2Bu(t), LDA, THF, ꢀ78 ꢁC, 2 h,
then ꢀ30 ꢁC, 15 h; (b) H2, 10% Pd/C, MeOH.
para-nitrophenyl urea derivatives, to be (7: 5R,6S) and
(8: 5S,6S).9
To illustrate the methodology towards the assembly of
muraymycin analogues, compounds 7 and 8 were inde-
pendently used for further transformations as outlined
in Schemes 2–4. The requisite linker aldehyde 11a–h was
preparedin two steps from the protectedamino acid
9a–h (R3=CBZ), which was coupledwith aminopro-
pane diethyl acetal to give an amide 10a–h, followedby
acidtreatment to form 11a–h (Scheme 2). The urea
moiety 17 was preparedin four steps from the FMOC
protectedarginine 12 by the following processes: (a)
esterification, 13, (b) removal of the FMOC group, 14,
(c) urea formation, 16, with valine benzyl ester (15), and
(d) regeneration of the acid (Scheme 3).
The antibacterial activity, as measuredby the minimal
inhibitory concentration (MIC, mg/mL), of 18a and 19a
as well as 21a and 22a was evaluatedin order to exam-
ine the effect of the protecting group (CBZ) on the
terminal amines (Table 1). Table 1 also illustrates the
stereochemical effect between 19a and 22a at the C5
position with regards to their antimicrobial activity.
Table 1 illustrates that both 19a and 22a were active in
the soluble peptidoglycan assay (SPG) with better
activity obtainedfor 22a (IC50 11 mg/mL). This suggests
Reductive amination of 7 with 11a using NaBH(OAc)3
resulted in the expected formation of the uridyl-dipep-
tide 18a. This reaction provedto be applicable to var-
ious l-leucine and(2 S,3S)-hydroxyleucine10 derivatives
(11b and 11c) exemplifiedby three combinations of R
1

A. Yamashita et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3345–3350
3347
a stereochemical preference for 5S. However, the MIC
values for 22a did not track with its SPG activity.
Compound 19a showedbetter MIC activity (4–8 mg/
mL) than 22a (8–>128 mg/mL) against various gram-
positive organisms.
The role of various protecting groups in 18 and 20 with
respect to their antimicrobial activities (Table 2) was
studied by employing a stepwise deprotection strategy.
This approach was appliedto leucine containing analo-
gue 18c (Scheme 5). Upon treatment of 18c with
nBu4NF, both TBS groups were removedto affordthe
corresponding diol 23c. The CBZ group in 23c was
readily removed by catalytic hydrogenation, to form the
free amine 24c. Hydrolysis of the tert-butyl ester in 23c,
followedby removal of the CBZ group, provideda
prototype of uracil protectedderivative 26c via 25c for
antimicrobial evaluation.
Basedon the above antibacterial data, a variety of ana-
logues 19a–h12 were preparedby coupling between
and 11a–h, andwere also evaluatedfor their anti-
bacterial activities (Table 2). A consistent trendwas
7
observedin the analogues carrying PMB, TBS and tert-
butyl protecting groups, in that the terminal unpro-
tectedamine forms possess variedantimicrobial activ-
ities (MICs 4–>128 mg/mL) against gram-positive
organisms, although their CBZ forms somewhat lost
their activities. Remarkably, this trendextends to the
aspartate andlysine analogues ( 19f and 19g).
Alternatively, the PMB group of the uracil ring in 18c
was removedby treatment with ceric ammonium nitrate
(CAN), to form the unprotecteduracil 27c. Catalytic
hydrogenation of 27c cleavedthe CBZ group, to form
Table 1. Comparison of in vitro activity for two diastereomers
Compd
18a
19a
21a
22a
1a
IC50 SPG (mg/mL)
32
32
10.7
10.7
—
Minimal inhibitory concentration (MIC) (mg/mL)
Gram-negative (10)
>128
>128
>128
64–128
32
>128
128
128
128
128
128
128
8
8–32
—
44
4
8
2
1
8
4
32
16
Candida albicans GC 4531
Staphylococcus aureus GC 4536
Staphylococcus aureus GC 1131
Staphylococcus aureus GC 2216
CNSa GC 4538
CNS GC 4538
CNS GC 4547
Enterococcus faecalis GC 842
Enterococcus faecalis GC 2242
Enterococcus faecalis GC 4555
128
>128
>128
>128
>128
>128
>128
128
4
8
4
4
4
8
4
8
4
128
128
128
128
128
128
64
>128
128
128
>128
>128
64
64
aCNS, coagulase-negative staphylococcus.
Table 2. In vitro activity of muraymycin analogues
CompdGram-negatives (10) Staphylococci (6) Enterococci (3)
18b
19b
18c
19c
18d
19d
18e
19e
18f
19f
18g
19g
18h
19h
20a
20b
20c
23c
24c
25c
26c
27c
28c
29c
30c
31c
32c
>128
>128
>128
>128
>128
>128
>128
64–>128
>128
128–>128
>128
128–>128
>128
>128
64–>128
>128
>128
128–>128
128–>128
128–>128
128–>128
128–>128
128–>128
128–>128
128–>128
128–>128
128–>128
>128
4–16
>128
4–64
128
>128
>128
4–8
>128
16–>128
>128
4–16
128
4–8
>128
4–16
128
16–>128
128
4–8
>128
4–32
>128
8
>128
>128
64–128
128–>128
>128
>128
>128
128
>128
>128
128–>128
>128
>128
>128
>128
1–2
>128
>128
>128
>128
128
128–>128
128
128
64–>128
2
128–>128
128–>128
128–>128
128–>128
Scheme 4. (a) 11a–h, NaBH(OAc)3, AcOH, THF; (b) H2, 10% Pd/C,
MeOH; (c) 17, HOBT, EDC, THF.

3348
A. Yamashita et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3345–3350
Scheme 6. (a) TFA, CH2Cl2; (b) H2, 10% Pd/C, MeOH, cat amount
AcOH.
Scheme 5. (a) nBu4NF, THF; (b) H2, 10% Pd/C, MeOH; (c) CAN,
MeCN, 65 ꢁC; (d) TFA, CH2Cl2.
Table 3. In vitro activity of muraymycin analogues
Organism
Compd
19b
19e
19g
28c
Minimal inhibitory concentration (MIC) (mg/mL)
Escherichia coli GC 4559
Escherichia coli GC 4560
Escherichia coli GC 3226
Serratia marcescens GC 4077
Morganella morganii GC 4531
Klebsiella pneumoniae GC 4534
Enterobacter cloacae GC 3783
Escherichia coli GC 2214
Pseudomonas aeruginosa GC 2214
Pseudomonas aeruginosa GC 4532
Candida albicans GC 3066
Staphylococcu aureus GC 4536
Staphylococcu aureus GC 1131
Staphylococcu aureus GC 2216
CNS GC 4538
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
>128
8
>128
32
8
>128
>128
64
128
>128
>128
>128
>128
>128
4
128
128
128
>128
>128
128
>128
>128
>128
8
>128
128
128
128
128
128
>128
128
32
1
1
2
1
4
4
4
4
8
4
4
8
8
CNS GC 4538
CNS GC 4547
4
16
8
4
8
16
2
2
Enterococcu faecalis GC 842
Enterococcu faecalis GC 2242
Enterococcu faecalis GC 4555
8
4
4
4
4
8
8
8
8
2
2
2
the free N-terminal analogue 28c. Treatment of 27c with
TFA not only cleavedboth TBS groups, but also
hydrolyzed the tert-butyl ester, to give 29c. The process
of deprotection of 20c followed the condition described
above. Catalytic hydrogenation cleaved both the benzyl
cleavedthe tert-butyl ester andthe two TBS groups
(Scheme 6).
Table 2 also demonstrates the SAR studies using the
leucine analogue 18c. When the TBS groups are
removed, the antibacterial activity was lost. However, in
andthe NO
groups, while treatment with TFA
2

A. Yamashita et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3345–3350
3349
(NCCLS).13 Muller-Hinton II broth (MHBII: BBL
Cockeysville, MD, USA) was the medium employed in
the testing procedures. Microtiter plates containing
serial dilutions of each antimicrobial agent were incu-
batedwith each organism to yieldthe appropriate den-
sity (105 CFU/mL) in a 100-mL final volume. The plates
Table 4. The inhibitory activity of muraymycin analogues against
MraY or MurG
Compd100 mg/mL 50 mg/mL 25 mg/mL 12.5 mg/mL 6.25 mg/mL
a
2a
+
+
+
+
ꢀ
+
ꢀ
+
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
19a
19b
19c
19d
19e
19f
19g
19h
22a
28c
32c
ꢁ
+
+
ꢀ
were incubatedfor 18–22 h at 35 C in ambient air. The
minimum inhibitory concentration (MIC) for all iso-
lates was defined as the lowest concentration of anti-
microbial agent that completely inhibits the growth of
the organism as detected by the unaided eye.
+
+
+
ꢀ
+
+
+
ꢀ
+
+
ꢀ
+
+
ꢀ
Determination of Lipid II Formation and Soluble
Peptidoglycan
a+ indicates reduced production of lipid II relative to the control
(judged by intensity) on the film, thus indicating inhibition of either
MraY of MurG. Since muraymycin A1 inhibitedthe production of
labeledlipidI andII due to inhibition of MraY, its closely related
analogues might be considered inhibitors of MraY.
The biological assay determining lipid II formation uti-
lizes S. epidermides membrane to catalyze the late steps
in cell wall biosynthesis including those performed by
MraY, the MurNAc pentapeptide translocase, and
MurG, the UDP-N-acetylglucosamyl transferase.
According to the reported method (J. Bacteriol. 1991,
173, 4625–4636), the formation of lipidII is assessed
using radiolabeled UDP-N-acetylglucosamine, S. epi-
dermides membranes, compound, UDP-MurNAc pen-
tapeptide, and [14C]-UDP-N-acetylglucosamine. The
reaction was incubatedat room temperature for 30 min
andwas terminatedby boiling in a water bath for 1 min.
The samples of each reactions were analyzedby separa-
tion using TLC. The plates were exposedto film, and
inhibition of lipidII formation was monitoredby com-
paring the area of the experimental sample to the area
of the control. The soluble peptidoglycan was deter-
minedusing a 96-well format to quantitate the produc-
tion of radioactive labeled peptidoglycan by S.
epidermides. The methodwas modifiedfrom that of
Boothby et al. (1971).
the unique case of 28c in which the PMB group in the
uracil ring was removed, the MIC value is restored upon
removal of the CBZ group. This result was consistent
with earlier observations.
More extensive in vitro activities (MICs) of selected
compounds (19b, 19e, 19g, and 28c) are shown in Table
3. Compound 28c demonstrated good antimicrobial
activity against various gram-positive organisms.
The inhibition of lipidII formation by muraymycin
analogues is summarizedin Table 4. With the exception
of 19d and 19h, most of the compounds of this type
inhibitedlipidII formation. This was most significant
for 19c, where the inhibition was observedas low as 25
mg/mL. These results corresponded well to the in vitro
antimicrobial tests, that 19a,b,c,e,g inhibitedlipidII
formation also showedmoderately goodantibacterial
activity against gram-positive isolates (MIC 4–16 mg/
mL). Compounds 19d and 19h did not inhibit lipid II
formation, andshowedpoor antibacterial activities
(MIC 16–>128 mg/mL). These results indicated that the
substitution at R1 in 19 has significant impact on the
inhibitory activity of target andbacteria. Overall, com-
pound 28c was the most potent one tested. It inhibited
lipidII formation at concentrations as low as 12.5 mg/
mL, although 28c has the same substitutions as com-
Acknowledgements
Authors thank analytical support provided by Dis-
covery Analytical Chemistry Department, Wyeth
Research, Pearl River, NY, andPrinceton, NJ, USA.
References and Notes
pound 19c with iso-propyl at R1 andhydrogen at R
.
2
1. Singh, M. P.; Greenstein, M. Curr. Opin. Drug Discov.
Develop. 2000, 3, 167.
2. Brodersen, D. E.; Clemons, W. M., Jr.; Carter, A. P.;
Morgan-Warren, R. J.; Wimberly, B. T.; Ramakrishnan, V.
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3. Healy, V. L.; Lessard, I. A. D.; Roper, D. I.; Knox, J. R.;
Walsh, C. T. Chem. Biol. 2000, 7, R109.
4. Isolation andstructure determination of muraymycin core
skeleton: McDonald, L. A.; Barbieri, L. R.; Carter, G. T.;
Lenoy, E.; Petersen, P. P.; Siegel, M. M.; Sign, G.; William-
son, R. T. J. Am. Chem. Soc. 2002, 124, 10260.
In conclusion, we have demonstrated the establishment
of a versatile methodology towards the synthesis of
various muraymycin analogues. The SAR in this series
establishes useful trends. Importantly, we have shown
that smaller fragments of the natural products provide
an excellent avenue for further development of potent
antimicrobial agents.
5. Biological activities of Muraymycins andderivatives were
reported: (a) Rasmussen, B. A.; McDonald, L. A.; Petersen, P.
P.; Rothstein, D.; Singh, G. 41st Interscience Conference on
Antimicrobial Agents andChemotherapy, Chcago, IL; Abstr
#1162. (b) McDonald, L. A.; Barbieri, L. R.; Carter, G. T.;
Method for In Vitro Antibiotics Evaluation
The in vitro activities of the antibiotics were determined
by the broth dilution method as recommended by the
National Committee for Clinical Laboratory Standards

3350
A. Yamashita et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3345–3350
Lenoy, E.; Petersen, P. P.; Siegel, M. M.; Singh, G. 41st
Interscience Conference on Antimicrobial Agents and
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Francisco, G. D.; McDonald, L. A.; Davis, R. A.; Singh, G.;
Yang, Y.; Mansour, T. S. Bioorg. Med. Chem. Lett. 2002, 12,
2341. (d) Singh, G.; Yang, Y.; Rasmussen, B. A.; Petersen, P.
P.; McDonald, L. A.; Yamashita, A.; Lin, Y.-I.; Norton, E.;
Francisco, G. D.; Li, Z.; Barbieri, L. R., 41st Interscience
Conference on Antimicrobial Agents andChemotherapy,
Chcago, IL; Abstr #1163.
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urea of 8 was preparedin a similar manner, andcrystals for
X-ray were obtainedfrom acetonitrile. The crystallographic
data for the para-nitrophenyl urea derivatives of 7 and 8 have
been deposited with the Cambridge Crystallographic Data Center,
12 Union Road, Cambridge CB2 1EW, UK, as supplementary
publication nos CCDC 199820 and199821, respectively.
10. (2S,3S)-3-Hydroxyleucine was prepared by using the lit-
erature procedure: Panek, J. S.; Masse, C. E. J. Org. Chem.
1998, 63, 2382.
11. Typical reaction conditions for formation of 18, 19, and
20 are as follows: Preparation of 18a: A mixture of 7 (230 mg,
0.319 mmol), 11a (108 mg, 0.35 mmol), NaBH(OAc)3 (135 mg,
0.64 mmol) and AcOH (3 drops) in anhydrous THF (5 mL)
was stirredat room temperature under nitrogen for 4 h, and
concentratedin vacuo. The resiude was chromatographed
(silica gel, 5% MeOH in CH2Cl2), to give 18a (174 mg, 54%)
as a pale-yellow solid. Preparation of 19a: A mixture of 18a
(146 mg, 0.144 mmol) was hydrogenated using 10% Pd/C (30
mg) in MeOH (3 mL) at room temperature under atomo-
spheric pressure. The catalyst was removedby filtration and
the filtrate was concentratedin vacuo, to give 19a as a white
amorphous solid(123 mg, 97%). Preparation of 20a: A solu-
tion of 17 (53 mg, 0.117 mmol), HOBT (16 mg, 0.117 mmol)
andEDC (22 mg, 0.117 mmol) in anhydrous THF (3 mL) was
stirredat room temperature for 30 min. To this mixture were
added a solution of 19a (98 mg, 0.111 mmol) in anhydrous
THF (2 mL) andHunig base (4 drops), andthe resulting mixture
was stirredat room temperature under nitrogen for 16 h. The
8. Banifi, L.; Cardani, S.; Potenza, D.; Scolastico, C. Tetra-
hedron 1987, 43, 2317.
9. Preparation of 7 and 8, and their para-nitrophenyl urea deri-
vatives: To a cooled( ꢀ78 ꢁC) solution of tert-butyl 2-(diben-
zylamino)propanoate (4.75 g, 15.23 mmol) in anhydrous THF
(60 mL) was added LDA (7.6 mL, 15.23 mmol, Aldrich)
dropwise under argon. After strring for 1 h at this condition, a
solution of 4 (4.5 g, 7.62 mmol) in THF (10 mL) was added via
syringe. The resulting mixture was stirredat ꢀ78 ꢁC for 2 h,
then at ꢀ35 ꢁC for 15 h, and quenched by addition of water.
The aqueous layer was extractedwith EtOAc, andthe com-
binedextracts were washedwith sat. brine solution, dried
(Na2SO4), andconcentratedin vacuo. The residue was chro-
reaction mixture was partitionedbetween EtOAc andH O. The
2
matographed(flash column, silica gel,
n-hexane/CH2Cl2/
organic layer was separated, and the aqueous layer was extrac-
tedwith EtOAc. The combinedextracts were washed(satd
brine), dried (Na2SO4), andconcentratedin vacuo. The pro-
duct was purified by chromatography (silica gel, 7% MeOH/
CH2Cl2), to give 20a as a white solid(84 mg, 58%).
12. Definition of R1 andR for a–h in compounds 18a–h,
2
19a–h, 20a–c, 21a, 22a, and 23c–32c is same as those in 11a–h
(Scheme 2).
13. NCCLS4. Methods for Dilution Antimicrobial Suscept-
ibility Test for Bacteria That Grow Aerobically; Approved
Standards: M7-A5, Vol. 20. National Committee for Clinical
Laboratory Standards, Wayne, PA, USA, 2000.
MeOH/ether=50/50/1/1), to give 6 (990 mg, 14%) and 5 (2.14
g, 31%). Further elution gave a mixture of two other diaster-
eomers (710 mg, 10%) andthe recovered 4 (1.6 g, 36%).
Compounds 5 and 6 were separately hydrogenated using 10%
Pd/C in MeOH, to give 7 and 8, respectively. A solution of 7
and para-nitrophenylisocyanate (1.2 mol equiv) in THF was
stirredat room temperature under nitrogen for 24 h. The
solution was concentratedin vacuo, andthe resiude was
chromatographed(flash column, silica gel, EtOAc/hexenes) to
give the corresponding urea. Crystals suitable for X-ray dif-
fraction were obtainedfrom methanol. The para-nitrophenyl