The synthesis and biological testing of bacilysin analogues

Article abstract of DOI:10.1007/s00726-013-1571-4

A series of compounds based on the structure of bacilysin were synthesised and tested for antibacterial activity. The key steps in the syntheses are the coupling of an iodide to a diketopiperazine (DKP) and mono-lactim ether scaffold, respectively. The diastereoselectivity of the coupling reactions was dependant on the scaffold, with selectivity for DKP of about 4:1 and mono-lactim ether exceeding 98:2. Subsequent elaboration of the compounds to give open chain dipeptides and DKPs that mimic the structure of bacilysin but substitute the epoxy ketone for a saturated or unsaturated ketone is described. Overall yield from coupling to final product was between 5 and 21 %, with the yield of the saturated products notably higher. The open chain dipeptides demonstrated moderate antibacterial and antifungal activity.

Full text of DOI:10.1007/s00726-013-1571-4

Amino Acids (2013) 45:1157–1168
DOI 10.1007/s00726-013-1571-4
ORIGINAL ARTICLE
The synthesis and biological testing of bacilysin analogues
•
Keith Robertson Cormac D. Murphy
•
Francesca Paradisi
Received: 30 April 2013 / Accepted: 23 July 2013 / Published online: 6 August 2013
Ó Springer-Verlag Wien 2013
Abstract A series of compounds based on the structure
of bacilysin were synthesised and tested for antibacterial
activity. The key steps in the syntheses are the coupling of
an iodide to a diketopiperazine (DKP) and mono-lactim
ether scaffold, respectively. The diastereoselectivity of the
coupling reactions was dependant on the scaffold, with
selectivity for DKP of about 4:1 and mono-lactim ether
exceeding 98:2. Subsequent elaboration of the compounds
to give open chain dipeptides and DKPs that mimic the
structure of bacilysin but substitute the epoxy ketone for a
saturated or unsaturated ketone is described. Overall yield
from coupling to final product was between 5 and 21 %,
with the yield of the saturated products notably higher. The
open chain dipeptides demonstrated moderate antibacterial
and antifungal activity.
antimicrobial drugs (Mathers et al. 2009). This is due in no
small part to the growing prevalence of pathogens that are
resistant to many of the current library of antibiotics
(Davies and Davies 2010). In spite of this, the develop-
mental pipelines of the major pharmaceutical companies
contain few antimicrobials (Spellberg et al. 2004; So et al.
2011). Classical screening programmes assaying for bio-
activity have high re-discovery rates, thus alternative
approaches for the development of new antibiotics are
required.
The dipeptide bacilysin (1) (originally termed bacillin
and occasionally known as tetaine) is one of the simplest
known antibiotics and is composed of alanine and the non-
proteinogenic amino acid anticapsin (Fig. 1). It was first
isolated by Foster and Woodruff (1946) from the soil
bacterium Bacillus subtilis and is active against a range of
bacteria and fungi. Bacilysin enters the cell via dipeptide
transport systems and is subsequently cleaved by host
peptidases to release the active agent anticapsin (Kenig
et al. 1976; Chmara et al. 1982), which is a competitive
irreversible inhibitor of glucosamine-6-phosphate (GlcN-6-
P) synthase (Perry and Abraham 1979; Kenig et al. 1976;
Chmara 1985; Chmara et al. 1984). One possible mecha-
nism of inhibition is shown in Scheme 1 and suggests that
opening of the epoxide is preceded by nucleophilic attack
by a cysteine residue on the active site the carbonyl of
anticapsin. The importance of the ketone moiety of anti-
capsin was demonstrated by inhibition studies with non-
stereospecific analogues of bacilysin and anticapsin lacking
the ketone moiety (Chmara et al. 1982), which were shown
to be much less effective inhibitors of GlcN-6-P synthase
than the natural compounds. Furthermore, studies in
aqueous environments have shown that epoxyketones such
as anticapsin tend to exist in equilibrium between the
ketone and the hydrated hemiacetal which would tend to
Keywords Anticapsin ꢀ Bacillus subtilis ꢀ Dipeptide ꢀ
Diketopiperazine
Introduction
Infectious diseases remain one of the leading causes of
death worldwide even with the widespread availability of
K. Robertson ꢀ F. Paradisi (&)
School of Chemistry and Chemical Biology, Centre for
Synthesis and Chemical Biology, University College Dublin,
Dublin, Ireland
e-mail: Francesca.paradisi@ucd.ie
C. D. Murphy (&)
School of Biomolecular and Biomedical Science, Centre for
Synthesis and Chemical Biology, University College Dublin,
Dublin, Ireland
e-mail: Cormac.d.murphy@ucd.ie
123

1158
K. Robertson et al.
Ph
O
Ph
targeting the enzyme have been reported (Fig. 1) (Woj-
ciechowski et al. 2005; Jedrzejczak et al. 2012; Bearne and
Blouin 2000; Gonzalez-Ibarra et al. 2010; Bates et al.
1966).
O
O
O
O
O
O
H
N
NH
CO2H
NH
Given the antimicrobial potential of bacilysin and rela-
ted compounds we report here a new synthetic strategy for
the production of bacilysin analogues (Fig. 2) and their
antimicrobial activities. Such analogues might be useful to
further explore the mechanism of GlcN-6-P synthase
inhibition, since no enzymological studies have yet been
conducted to investigate the importance of the epoxide.
NH2
HO2C
O
H2N
H2N
CO2H
1
2
3
Fig. 1 The structure of bacilysin (1) and N3-oxoacyl derivatives of
1-2,3-diaminopropanoic acid (2 and 3), known inhibitors of GlcN-6-P
synthase
Results and discussion
H
S
S
O
O
HO
S
There are a number of synthetic routes to bacilysin and
anticapsin available in the literature, with the first enan-
tioselective versions published independently by Wild
(1994) and Baldwin et al. (1995). Our initial synthesis
planned to make use of a masked a,b–unsaturated alkyl-
ating agent in conjunction with either a diketopiperazine
(DKP) or mono-lactim ether (MLE) depending on the
desired product. The initial strategy was to prepare this
alkylating agent via a Diels–Alder reaction of a Dani-
shefsky-type diene with acrolein, which would be activated
to the iodide in a Finkelstein reaction. However, the
iodination of (4-(tert-butyldimethylsilyloxy)-2-methox-
ycyclohex-3-enyl) methyl methanesulfonate in refluxing
acetone furnished 4-methylenecyclohex-2-enone instead of
the expected iodide (Lestini et al. 2012). An alternative
strategy was designed utilising a saturated alkylating agent
similar to the synthesis described by Wild (1994)
(Scheme 2). Ethyl 4-hydroxycyclohexanecarboxylate 8 as
a mixture of cis and trans isomers was commercially
available; the hydroxyl group was silylated using tert-
butyldimethylsilyl triflate and triethylamine in THF.
Cleavage of the ester to give a hydroxyl group was
accomplished using lithium aluminium hydride in THF at
0 °C. The exposed hydroxyl group of 10 was subsequently
activated to the iodide 12, via the mesylate. This was
accomplished in good yield and thus the alkylating agent
was available on a multi-gram scale for use in further
synthesis.
O
O
OH
NH2
CO2H
NH2
NH2
CO2H
CO2H
Scheme 1 One possible mechanism of anticapsin inhibition of GlcN-
6-P synthase by covalently bonding to the sulphydryl group in the
glutamine binding site of GlcN-6-P synthase
O
O
O
O
O
O
HN
HN
NH
NH
5
4
O
O
O
O
OH
OH
N
N
H
H
NH2
O
NH2
O
6
7
The MLE was synthesised as previously reported
(Paradisi et al. 2001); however, to allow for milder de-
protecting conditions of the amide, para-methoxybenzyl
chloride instead of benzyl chloride was employed in the
first step (Scheme 3). The cleavage of the benzyl group
could be performed under Birch conditions which would
reduce the unsaturated ketone. Indeed, as was the case for
similar syntheses (Wild 1994; Baldwin et al. 1995), it was
observed that basic conditions promoted a 1,4-intramo-
lecular addition of the amide onto the enone system.
Fig. 2 The bacilysin analogues synthesised in this study
lower the electrophilicity of the epoxyketone and favour
the proposed mechanism (Wipf et al. 1998; Baldwin et al.
1995). The lack of GlcN-6-P brought about by the inhibi-
tion of GlcN-6-P synthase is tolerated by mammals, at
least in the short term, but is sufficiently detrimental to
microbes that a number of anti-microbial compounds
123

The synthesis and biological testing
1159
O
OH
OTBDMS
OTBDMS
OH
PhMP
N
NH2
i)
N
PMPh
i)
ii)
O
O
20
19
O
O
O
O
Scheme 4 i (a) Chloroacetyl chloride, CH2Cl2, NaOH(aq) 0 °C
(b) Triethylbenzylammonium chloride
8
9
10
was trans to the iso-propyl group in a 98:2 ratio (R:S). The
DKP gave the alkylated group in an S configuration with a
ratio of 81:19. These values are comparable to those
reported in the literature (O’Reilly et al. 2009; Davies et al.
2007). The silyl protecting group of the DKP adduct was
cleaved with acetic acid; however, for the MLE adduct
TBAF was used to avoid the cleavage of the lactim bond,
which occurs under mild acidic conditions. The exposed
alcohol moiety was then oxidised under Swern conditions
to give the ketones 22 and 25 in good yield. The para-
methoxy benzyl protecting groups were cleaved using
CAN in acetonitrile/water. The best results for this
deprotection were obtained with the minimal requirements
of two equivalents of CAN, as excess unreacted reagent
proved slightly difficult to purify from the product. Puri-
fication after deprotection yielded 4 in the case of the DKP
adduct, whereas to generate the dipeptide 6, a partial
purification had to be followed by a mild acidic hydrolysis
and then a more stringent purification. The unsaturated
ketone moiety was generated in both cases starting from 22
or 25 by use of an iodic acid-dimethylsulphoxide complex
as described by Nicolaou et al. (2002). The advantage of
this technique was that it allowed the enone system to be
generated in a single step from the ketone, albeit racemi-
cally. 23 and 26 were then subjected to the deprotection
and purification or deprotection, partial purification, acidic
hydrolysis and purification as outlined previously to give 5
and 7, both as racemic mixtures. The yields were signifi-
cantly lower than for the saturated analogues, probably as a
iii)
OTBDMS
OTBDMS
iv)
I
OMs
11
12
Scheme 2 i TBDMSOTf, Et3N, CH2Cl2, 0 °C ii LiAlH4, THF, 0 °C
iii MsCl, Et3N, CH2Cl2 iv NaI, acetone
By contrast, the para-methoxybenzyl protecting group was
easily cleaved with cerium ammonium nitrate which left
the enone moiety intact. Despite special care taken both in
the work-up and in subsequent steps to avoid basic con-
ditions, the yield was lower than expected and may be
partially justified by the propensity of the enone system to
undergo addition.
The DKP was synthesised in a one pot reaction as pre-
viously described by O’Reilly et al. (2009) (Scheme 4).
Again para-methoxy benzyl-methyl amine was used
instead of the classic benzyl-methyl amine. The coupling
of 12 to either 18 or 20 was accomplished by treating with
lithium hexamethyldisilyl azide at -10 °C in THF and
subsequently alkylating with 12 at -78 °C (Scheme 5). As
expected, the newly generated chiral centre of the MLE
Scheme 3 i SOCl2, MeOH,
O
O
0 °C ii PMBCl, pyridine,
CH2Cl2, 0 °C iii ClCOCH2Cl,
Et3N, CH2Cl2, 0 °C iv NH3,
EtOH v Et3OBF4, CH2Cl2
O
O
i)
ii)
iii)
OMe
Cl
OMe
OH
OMe
N
PMB
NH
NH2
NH2
PMB
O
13
14
15
16
iv)
OEt
O
v)
N
NH
N
N
PMB
PMB
O
O
18
17
123

1160
K. Robertson et al.
i)
i)
12
18
21
12
20
24
ii)
iii)
ii)
iii)
iv)
vi)
6
22
4
25
v)
v)
iv)
vi)
7
23
5
26
Scheme 5 i (a) 18/20, LHMDS, THF, -10 °C (b) 12, THF, -78 °C ii TBAF, THF or CH3CO2H, THF, H2O iii Oxalyl chloride, DMSO, Et3N,
CH2Cl2, -78 °C iv (a) CAN, CH3CN, H2O (b) 0.5 M HCl, acetone, 40 °C v HIO3, DMSO, cyclohexene, 50 °C vi CAN, CH3CN, H2O
result of the aforementioned intramolecular addition of the
amide onto the enone system, which could be avoided by
reducing the unsaturated ketone to the vinyl alcohol before
the deprotection step and then oxidising it back to the
enone as one of the final steps (Wild 1994; Baldwin et al.
1995). However, this would have added an additional two
steps to the overall route and in this case the drop in yield
was tolerated.
inhibitory concentrations for all compounds were greater
than 3.2 lg mL-1 and no effect was observed on the
growth of the bacteria. Although no authentic bacilysin was
available for direct comparison, previous bioassay
employing comparable methods (Kenig and Abraham
1976) indicates that 6 and 7 are weaker antimicrobial
agents than the natural product.
Initial screening for antimicrobial activity of 4–7 was
carried out using the Kirby Bauer disc diffusion assay
against standard laboratory strains of Staphylococcus aur-
eus, Escherichia coli, Saccharomyces cerevisiae and
Candida albicans. The open chain dipeptides 6 and 7
showed activity (zones of clearing, 6–8 mm) against both
strains of yeast and to a lesser extent against the Gram
positive S. aureus, but not the Gram negative E. coli.
However, the DKPs 4 and 5 showed no sign of activity
against any of the microbes. Additional testing against a
range of antibiotic-resistant bacteria and clinical isolates
was conducted, but it was found that the minimum
Conclusion
Novel bacilysin analogues were prepared via coupling of
an iodinated alkalyating reagent with either a DKP or
mono-lactim ether. The antimicrobial activity of the open
chain analogues was modest, suggesting that the epoxide
moiety is significant in the inhibition of GlcN-6-P synthase.
The DKPs had no antimicrobial activity possibly because
of poor uptake into cells and/or incomplete hydrolysis of
the compounds to release the anticapsin-like warhead.
Nevertheless, the synthetic strategy might be employed to
123

The synthesis and biological testing
1161
generate further bacilysin-like compounds with improved
biological properties.
((CH2)2CH(OTBDMS), 26.0 (SiC(CH3)3), 23.6 ((CH2)2CH
(CH2OEt)), 18.3 (SiC(CH3)3), 14.4 (CH2CH3), -4.7
(Si(CH3)2). HR-MS: m/z Calcd for C15H30O3NaSi
(M ? Na)?: 309.1862; found 309.1876.
Experimental
Minor isomer: 1H NMR (500 MHz, Chloroform-d) d
4.12–4.07 (m, 2H, overlap with major isomer, CH2CH3),
3.60–3.53 (m, 1H, CHOSi), 2.25–2.18 (m, 1H, CHCO2),
1.99–1.86 (m, 3H, overlap with major isomer), 1.69–1.61
(m, 3H, overlap with major isomer), 1.52–1.42 (m, 2H,
overlap with major isomer), 1.24–1.22 (m, 3H, overlap
with major isomer), 0.88 (s, 9H, SiC(CH3)3), 0.05 (s, 6H,
Si(CH3)2). 13C NMR (126 MHz, Chloroform-d) d 175.8 9
(CO2Et), 70.7 (HCOTBDMS), 60.3 (OCH2CH3), 42.5
(CHCO2Et), 35.0 ((CH2)2CH(OTBDMS), 27.4 ((CH2)2
CH(CH2OEt)), 26.0 (SiC(CH3)3), 18.3 (SiC(CH3)3), 14.4
(CH2CH3), -4.5 (Si(CH3)2).
All chemicals were purchased from Sigma-Aldrich unless
otherwise stated. Dry solvents were obtained from a Pu-
resol Grubbs Speciality Chemicals and Stream. Where
necessary reactions were performed under a nitrogen
atmosphere, using oven-dried glassware. Oxygen-free
nitrogen was obtained from BOC gases and used without
further drying.
1H and 13C-NMR spectra were recorded on a Varian
NMR System 500 MHz, Varian NMR System 400 MHz or
Varian NMR System 300 MHz System spectrometer. High
resolution mass spectra were obtained using a Waters/Mi-
cromass instrument. Removal of solvent under reduced
pressure refers to the removal of solvent on a Bu¨chi rotary
evaporator with an integrated vacuum pump. Thin-layer
chromatography (TLC) was carried out on Merck alumin-
ium backed 60 F254 silica gel. Purifications were carried
out using forced flow chromatography on Merck silica gel
60 (0.040–0.063 nm) or Merck aluminium oxide, 90,
standardised.
(4-(Tert-butyldimethylsilyloxy)cyclohexyl)methanol
(10)
9 (2.72 g, 9.5 mmol) was dissolved in dry THF (22 mL)
under nitrogen and cooled to 0 °C. Lithium aluminium
hydride (0.43 g, 11.3 mmol) was added in portions. The
mixture was allowed to warm to room temperature and
stirred for 5 h when TLC showed completion of reaction.
The reaction mixture was quenched by careful, slow
addition of ice cold water (5 mL). The mixture was acid-
ified using 1 M HCl to pH 2. The mixture was filtered and
the layers separated. The aqueous layer was extracted a
further three times with CH2Cl2 (35 mL). The combined
organic layer was washed with saturated NaHCO3 solution
(20 mL), water (20 mL) and brine (20 mL). The organic
layer was then dried with MgSO4 and solvent was removed
under reduced pressure to give 10 as a pale yellow oil in
94 % yield (2.18 g).
Major isomer: 1H NMR (500 MHz, Chloroform-d) d
3.90 (m, 1H, CHOSi), 3.12 (d, J = 5.7 Hz, 2H, CH2OH),
1.89–0.90 (9H, aliphatic H, overlap with minor isomer,
0.89 (s, 9H, (CH3)3CSi), 0.03 (s, 6H, (CH3)2Si). 13C NMR
(126 MHz, CDCl3) d 68.4(CHOTBDMS), 67.1 (CH2OH),
39.7 (CHCH2OH), 33.0 ((CH2)2CH(OTBDMS), 26.0
(SiC(CH3)3), 23.6, 18.3 (SiC(CH3)3), -4.7 (Si(CH3)2).
HR-MS: m/z Calcd for C13H28O2NaSi (M ? Na)?:
267.1756; found: 267.1760.
Ethyl 4-(tert-butyldimethylsilyloxy)cyclohexane-
carboxylate(mixture of trans and cis isomers) (9)
8 (1.62 mL, 10 mmol) was dissolved in dry CH2Cl2
˚
(35 mL) under nitrogen with activated 4 A molecular
sieves. The stirred solution was cooled to 0 °C and triethyl
amine (2.8 mL, 20 mmol) was added followed by tert-
butyldimethylsilyl triflate (2.52 mL, 11 mmol) dropwise.
The mixture was allowed to warm to room temperature and
stirred for 12 h when TLC showed consumption of starting
material. The reaction was filtered and diluted with CH2Cl2
(30 mL). The organic layer was washed with saturated
NaHCO3 solution (25 mL), water (25 mL), brine (25 mL)
and dried with Na2SO4, and solvent was removed under
reduced pressure. The resulting yellow residue was purified
on silica using 4:1 cyclohexane:EtOAc as an eluent
yielding 2.72 g ethyl 4-(tert-butyldimethylsilyloxy)cyclo-
hexanecarboxylate as a pale yellow oil (95 % yield).
Major isomer: 1H NMR (500 MHz, Chloroform-d) d
4.17–4.07 (m, 2H, overlap with minor isomer CH2CH3), 3.89
(p, J = 2.1 Hz, 1H, CHOSi), 2.32–2.25 (m, 1H, CHCO2),
1.99–1.87 (m, 3H, overlap with minor isomer), 1.69–1.61
(m, 3H, overlap with minor isomer), 1.54–1.41 (m, 2H),
1.26–1.24 (m, 3H, CH2CH3 overlap with minor isomer), 0.89
(s, 9H, SiC(CH3)3), 0.03 (s, 6H Si(CH3)2). 13C NMR
(126 MHz, Chloroform-d) d 175.9 (CO2Et), 66.9 (HCOT-
BDMS), 60.2 (OCH2CH3), 42.3 (CHCO2Et), 33.0
Minor isomer: 1H NMR (500 MHz, CDCl3) d 3.56–3.49
(m, 1H, CHOSi), 3.44 (d, J = 6.3 Hz, 2H, CH2OH),
1.92–0.92 (m, 9H, aliphatic H, overlap with major isomer),
0.88 (s, 9H, (CH3)3CSi, overlap with major isomer), 0.03
(s, 6H, (CH3)2Si, overlap with major isomer). 13C NMR
(126 MHz, CDCl3) d 71.9 (CHOTBDMS), 68.3 (CH2OH),
39.8 (CHCH2OH), 35.5 ((CH2)2CH(OTBDMS), 27.9
((CH2)2CH(CH2OH)), 26.1 (SiC(CH3)3), 18.4 (SiC(CH3)3),
-4.4 (Si(CH3)2).
123

1162
K. Robertson et al.
(4-(Tert-butyldimethylsilyloxy)cyclohexyl)methyl
methanesulfonate (11)
39.7, 33.1, 27.4, 26.0, 18.2, 15.6, -4.7 (Si(CH3)(CH3),
(Si(CH3)(CH3)). Elemental analysis for C13H27IOSi
required: C 44.1, H 7.7; found: C 44.0, H 7.5.
10 (2.18 g, 8.9 mmol) was dissolved in dry CH2Cl2
˚
(20 mL) under nitrogen with activated 4 A molecular
(S)-Valine methyl ester (14)
sieves. The solution was cooled to 0 °C and triethyl amine
(3.12 mL, 22.25 mmol) was added. Methanesulphonyl
chloride (0.8 mL, 9.8 mmol) was added dropwise to the
mixture. The reaction was allowed to warm to room tem-
perature and stirred for 72 h when TLC showed disap-
pearance of starting material. The reaction was worked up
by filtering to remove molecular sieves and washing the
reaction mixture with saturated NaHCO3 solution (20 mL),
water (20 mL) and brine (20 mL). The organic layer was
then dried with MgSO4 and solvent was removed under
reduced pressure to give 2.71 g (94 % yield) of 11 as a pale
yellow oil. No further purification was necessary.
Major isomer: 1H NMR (500 MHz, Chloroform-d) d
4.04 (d, J = 6.9 Hz, 2H, CH2OMs), 3.99–3.96 (m, 1H,
CHOTBDMS), 2.99 (s, 3H, SCH3), 1.93–1.78 (m, 2H),
1.72–1.62 (m, 2H), 1.48–1.43 (m, 3H), 1.31 (qd, 13.6,
3.9 Hz, 1H), 1.07 (qd, J = 13.5, 3.6 Hz, 1H), 0.89 (s, 9H,
SiC(CH3)3), 0.03 (s, 6H, Si(CH3)2). 13C NMR (126 MHz,
Chloroform-d) d 74.7 (CHOTBDMS), 66.4 (CH2OMs),
37.4 (CHCH2OMs), 36.6 ((CH2)2CHOTBDMS), 32.6
((CH2)2CHCH2OMs), 26.0 (SiC(CH3)3), 23.2 (SCH3), 18.2
(SiC(CH3)3), -4.7 (Si(CH3)2). HR-MS: m/z Calcd for
C14H30O4NaSSi (M ? Na)?: 345.1532; found 345.1543.
Minor isomer: 1H NMR (500 MHz, Chloroform-d) d
4.03 (d, J = 6.6 Hz, 2H), 3.53 (m, 1H), 4.04 (s, 3H,
overlap with major isomer), 1.95–1.03 (m, 9H, overlap
with major isomer), 0.88 (s, 9H), 0.05 (s, 6H).
L-Valine (13) (11.9 g, 0.1 mol) was dissolved in dry
methanol (100 mL) and cooled to 0 °C. To the stirred
solution, thionyl chloride (21 mL, 0.29 mol) was added
slowly and the mixture was stirred until disappearance of
the starting material was observed by TLC. The reaction
was worked up by removal of solvent under reduced
pressure and washed with Et2O (five times) to give the
hydrochloride salt as a white solid. (14.9 g, 89 % yield).
This was dissolved in a mixture of NaHCO3 solution
(75 mL) and EtOAc (75 mL). The layers were separated
and the aqueous layer was extracted with EtOAc (50 mL)
twice more and the combined organic layer was washed
with brine (50 mL), dried over Na2SO4 filtered and solvent
was removed under high vacuum directly before the next
step to minimise decomposition.
1H NMR (500 MHz, DMSO-d6) d 8.39 (br s, 3H, (NH3),
3.86 (d, J = 4.9 Hz, 1H, CHCHCH3), 3.76 (s, 3H, OCH3),
2.20–2.13 (m, 1H, CH(CH3)2), 0.98 (d, J = 6.9 Hz, 3H,
CH3CHCH3), 0.94 (d, J = 6.8 Hz, 3H, CH3CHCH3). 13C
NMR (126 MHz, DMSO) d 169.3 (MeOC=O), 57.3
(CHCH(CH3)2), 52.6 (OCH3), 29.3 (CH(CH3)2), 18.2
(CH3CHCH3), 17.6 (CH3CHCH3). HR-MS: m/z Calcd for
C6H14O2N (M ? H)?: 132.1025, found: 132.1031.
(S)-Methyl 2-(4-methoxybenzylamino)-3-
methylbutanoate (15)
Tert-butyl(4-(iodomethyl)cyclohexyloxy)
dimethylsilane (12)
14 (4.37 g, 33.2 mmol) was dissolved in dry CH2Cl2
(40 mL) and cooled to 0 °C. Pyridine (2.95 mL,
36.5 mmol) was added and the solution was stirred for
5 min. Para-methoxybenzylchloride (4.9 mL, 36.5 mmol)
was added slowly and the mixture was allowed to warm to
room temperature and stirred for a further 12 h. The
reaction was worked up by the addition of water (50 mL)
and the layers were separated and the aqueous layer was
extracted with CH2Cl2 (40 mL) a further two times. The
combined organic layer was washed with brine (40 mL)
dried over Na2SO4 and the solvent was removed under
reduced pressure. The crude product was purified on silica
gel using ethyl acetate: cyclohexane as an eluent to give the
pure product in 80 % yield (6.67 g).
11 (2.71 g, 8.4 mmol) and sodium iodide (3.15 g, 21 mmol)
were dissolved in acetone (30 mL) and refluxed for 12 h
when TLC showed disappearance of starting material. The
reaction was worked up by removal of the solvent under
reduced pressure; the residue was dissolved in a mixture of
CH2Cl2 (35 mL) and water (35 mL). The layers were sepa-
rated and the aqueous layer was extracted a further three
times with CH2Cl2 (15 mL). The organic layers were com-
bined, washed with brine (25 mL) dried with MgSO4 and
solvent was removed under reduced pressure to give a yellow
oil. The crude mixture was purified on silica using a 5:1
mixture of cyclohexane: ethyl acetate as an eluent to give 12
as a clear non-viscous oil in 83 % yield (2.47 g).
1H NMR (500 MHz, Chloroform-d)
d 7.25 (d,
J = 8.6 Hz, 2H, ArH), 6.85 (d, J = 8.7 Hz, 2H, ArH),
3.79 (s, 3H, OCH3), 3.76 (d, J = 12.7 Hz, 1H, ArCHH),
3.72 (s, 3H, OCH3), 3.52 (d, J = 12.8 Hz, 1H, ArCHH),
3.01 (d, J = 6.2 Hz, 1H, NHCHC=O), 1.91 (m, 1H,
CH(CH3)2), 1.70 (s, 1H, NH), 0.94 (d, J = 6.8 Hz, 3H,
1H NMR (500 MHz, Chloroform-d) d 3.92–3.87 (m, 1H,
CHCH2I), 3.12 (d, J = 5.7 Hz, 2H, CH2I), 1.68–1.58 (m,
4H), 1.51–1.41 (m, 4H), 0.89 (s, 9H, (CH3)3C), 0.03 (s, 6H,
Si(CH3)2). 13C NMR (126 MHz, Chloroform-d) d 66.4,
123

The synthesis and biological testing
1163
CHCH3), 0.92 (d, J = 6.7 Hz, 3H, CHCH3). 13C NMR
(126 MHz, Chloroform-d) d 175.9 (OC=O), 158.8 (Ar),
132.4 (Ar), 129.5 (Ar), 113.8 (Ar), 66.6 (NHCH), 55.4
(OCH3), 52.1 (CH2), 51.5 (OCH3), 31.8 (CH(CH3)2), 19.4
(CH(CH3)(CH3)), 18.81 (CH(CH3)(CH3)). HR-MS: m/z
Calcd for C14H21O3NNa (M ? Na)?: 274.1419; found:
6.00 (s, 1H NH), 5.37 (d, J = 14.7 Hz, 1H, NHCHHC=O),
4.13 (d, J = 17.5 Hz, 1H, NCHHAr), 3.96 (dd, J = 17.4,
4.3 Hz, 1H, NCHHAr), 3.86 (d, J = 14.5 Hz, 1H,
NHCHHC=O), 3.80 (s, 3H, OCH3), 3.67 (d, J = 4.9 Hz,
1H, CHCH(CH3)2), 2.25 (m, 1H, CH(CH3)2), 1.12 (d,
J = 6.9 Hz, 3H, CH(CH3)(CH3)), 1.04 (d, J = 6.9 Hz, 3H,
CH(CH3)(CH3)). 13C NMR (126 MHz, Chloroform-d) d
167.5 (NHC=O), 164.5 (NC=O), 159.6 (ArO), 129.7 (Ar),
127.7 (Ar), 114.5 (ArCH2), 64.5 (CHCH(CH3)2), 55.5
(OCH3), 48.0 (NCH2Ar), 45.6 (O=CCHHN), 32.1
(CH(CH3)2), 20.0 (CHCH3), 17.9 (CHCH3). HR-MS: m/z
Calcd for C15H20N2O3Na (M ? Na)?: 299.1372; found
20
274.1408. ½aꢁD ¼ ꢂ39ꢃðc ¼ 0:96; CHCl3Þ.
(S)-Methyl 2-(2-chloro-N-(4-
methoxybenzyl)acetamido)-3-methylbutanoate (16)
15 (2.94 g, 11.7 mmol) was dissolved in dry CH2Cl2
(25 mL) and cooled to 0 °C. Triethyl amine (2 mL,
14.1 mmol) was added to the stirred solution, chloroacetyl
chloride (1.1 mL, 14.1 mmol) was added dropwise and the
reaction was stirred for additional 12 h. The reaction was
worked up by the addition of water (30 mL), the layers
were separated and the aqueous layer was extracted with
(40 mL) a further two times. The combined organic layer
was washed with brine (40 mL) dried over Na2SO4 and the
solvent was removed under reduced pressure. The crude
product was purified on silica gel using ethyl acetate:
cyclohexane as an eluent to give the pure product in 89 %
yield (3.4 g).
20
299.1380.½aꢁD ¼ ꢂ14:2ꢃðc ¼ 1:02; CHCl3Þ.
(S)-5-Ethoxy-6-isopropyl-1-(4-methoxybenzyl)-1,6-
dihydropyrazin-2(3H)-one (18)
An oven-dried three-necked flask with a stopcock sidearm
was equipped with a condenser under nitrogen. Boron
fluoride etherate (1.41 mL, 11.2 mmol) was dissolved in
the flask in dry Et2O (50 mL). To the stirred solution epi-
chlorohydrin (0.57 mL, 7.01 mmol) was added at a rate to
maintain boiling. The reaction mixture was refluxed for a
further hour and then allowed to stand at room temperature
overnight. Pale yellow solid had formed which was col-
lected by filtering off the solvent under nitrogen and washed
a further three times with dry diethyl ether (3 9 35 mL). 17
(1.55 g, 5.6 mmol) dissolved in CH2Cl2 (50 mL) was added
and the reaction was stirred for a further 12 h. The reaction
was worked up by addition of aqueous KHCO3 solution
(40 mL). The layers were separated and the aqueous layer
was extracted a further two times with CH2Cl2 (50 mL).
The combined organic layer was washed with brine, dried
over Na2SO4 and the solvent was removed under reduced
pressure. The resulting orange residue was purified by flash
chromatography on silica using 1:1 EtOAc: cyclohexane as
an eluent. Product was recovered as a light orange, viscous
oil (1.28 g, 60 % yield).
1H NMR (400 MHz, Chloroform-d)
d 7.07 (d,
J = 8.2 Hz, 2H, ArH), 6.86 (d, J = 8.2 Hz, 2H, ArH),
4.77 (d, J = 10.3 Hz, 1H, NCH), 4.64 (s, 2H, CH2Ar), 3.78
(s, 3H, OCH3), 3.48 (s, 2H, CH2Cl), 2.44–2.29 (m, 1H,
CH(CH3)2), 0.98 (d, J = 6.4 Hz, 3H, CH3CHCH3), 0.92
(d, J = 6.7 Hz, 3H, CH3CHCH3). 13C NMR (100 MHz,
Chloroform-d) d 171.2 (O=COMe), 162.2 (O=CCH2Cl),
159.5 (ArOMe), 129.4 (Ar), 128.7 (Ar), 114.9 (Ar), 64.3
(CHCH(CH3)2), 56.0 (ArOCH3), 51.7 (O=COCH3), 46.4
(CH2Ar), 40.9 (CH2Cl), 28.3 (CH3CHCH3), 19.5
(CH3CHCH3), 19.0 (CH3CHCH3). HR-MS: m/z Calcd for
C16H22O4NClNa (M ? Na)?: 350.1135; found 350.1135.
20
½aꢁD ¼ ꢂ44:6ꢃðc ¼ 0:98; CHCl3Þ.
1H NMR (400 MHz, Chloroform-d) d 7.20–7.10 (m, 2H,
ArH), 6.86 (d, J = 8.6 Hz, 2H, ArH), 5.39 (d,
J = 14.8 Hz, 1H, O=CCHHN), 4.16 (dd, J = 17.4, 1.3 Hz,
2H, NCH2Ar), 4.13–4.08 (m, 2H, CH2CH3), 3.84 (d,
J = 14.9 Hz, 1H, O=CCHHN), 3.79 (s, 3H, OCH3), 3.66
(m, 1H, CHCH(CH3)2), 2.24–2.14 (m, 1H, CH(CH3)2),
1.23 (t, J = 7.1 Hz, 3H, CH2CH3), 1.03 (d, J = 7.0 Hz,
3H, CHCH3), 0.91 (d, J = 6.9 Hz, 3H, CHCH3). 13C NMR
(101 MHz, Chloroform-d) d 168.2 (C=O), 160.6 (N=CO,
159.3 (ArO), 129.7 (Ar), 128.1 (Ar), 114.3 (Ar), 61.5
(CH2CH3), 61.2 (CHCH(CH3)2), 55.4 (OCH3), 51.1
(NCH2Ar), 46.6 (O=CCHHN), 31.9 (CH(CH3)2), 20.1
(CHCH3), 17.6(CHCH3), 14.4 (CH2CH3). HR-MS: m/z
(S)-6-Isopropyl-1-(4-methoxybenzyl)piperazine-2,5-
dione (17)
16 (3.63 g, 11.1 mmol) was stirred in a saturated solution
of ammonia in ethanol (100 mL) until starting material was
consumed (observed by TLC, 10 h). Ethanolic ammonia
was removed under reduced pressure and the residue was
washed with ice cold Et2O. The precipitate was purified on
silica using 4:1 cyclohexane: EtOAc as an eluent, and
2.99 g of product was recovered as a white solid. (97 %
yield).
1H NMR (500 MHz, Chloroform-d)
d 7.16 (d,
J = 8.6 Hz, 2H, ArH), 6.86 (d, J = 8.7 Hz, 2H, ArH),
123

1164
K. Robertson et al.
Calcd for C17H25N2O3 (M ? H)?: 305.1865; found:
EtOAc (50 mL). The combined organic layer was washed
with brine (50 mL), dried over Na2SO4 and the solvent was
removed under vacuum to give crude 21 as a brown resi-
due. Crude 21 (1.16 g, 2.17 mmol) was immediately dis-
solved in THF (10 mL). A 1 M solution of TBAF in THF
(5.24 mL, 5.24 mmol) was added and the mixture was
stirred at room temperature for 8 h when TLC showed
consumption of the starting material. The solvent was
removed under vacuum and the residue was dissolved in
CH2Cl2 (50 mL) and washed with deionised water (50 mL)
and the layers were separated. The aqueous layer was
extracted a further two times with CH2Cl2 (50 mL) and the
combined organic layer was washed with brine (35 mL),
dried over Na2SO4 and the solvent was removed under
reduced pressure to give an orange oil. Oxalyl chloride
(0.22 mL, 2.6 mmol) was dissolved in dry CH2Cl2
(12 mL) at -78 °C under nitrogen, dry DMSO (0.41 mL,
5.75 mmol) was slowly added and the reaction mixture was
stirred for 5 min. The orange oil which had been dried
thoroughly on a high vacuum pump was dissolved in
CH2Cl2 (5 mL) and added slowly to the reaction mixture
and was stirred for a further hour, taking care to keep the
temperature at -78 °C. Triethylamine (1.47 mL,
10.43 mmol) was added and the reaction was allowed to
warm to room temperature. The mixture was diluted with
CH2Cl2 (50 mL) and poured into aqueous NaHCO3 solu-
tion (35 mL). The layers were separated and the aqueous
layer was extracted a further two times with CH2Cl2
(35 mL). The combined organic layer was washed with
aqueous NaHCO3 solution (35 mL), water (35 mL), brine
(35 mL), dried over Na2SO4 and the solvent was removed
under reduced pressure to give an orange oil. Purification
was carried out on silica gel using 1:1 cyclohexane, ethyl
acetate as an eluent, and 530 mg of product was recovered
as a light orange viscous oil (39 % combined yield).
20
305.1852. ½aꢁD ¼ ꢂ29:5ꢃðc ¼ 1:02; CHCl3Þ.
1,4-Bis((S)-1-(4-methoxyphenyl)ethyl)piperazine-2,
5-dione (20)
(S)-1-(4-Methoxyphenyl)ethanamine (19) (40 mmol, 5.9 mL)
was dissolved in dichloromethane (80 mL) and cooled to
0 °C. To the stirred solution, 50 % (w/v) sodium hydroxide
solution (320 mmol, 25.6 mL) was added slowly. Chloro-
acetyl chloride (40 mmol, 3.2 mL) was added slowly to the
stirred mixture. The mixture was stirred for 12 h when TLC
showed that the amine had been consumed. N-benzyl-N,N,N-
triethylethanaminium chloride (4 mmol, 1.02 g) was added
portion-wise over a 48 h period. Work-up was performed by
separating the organic and aqueous layers and evaporating the
organic layer under reduced pressure. The residue was dis-
solved in EtOAc (75 mL), washed with 1 M HCl (25 mL),
water (35 mL), brine (35 mL) dried over MgSO4 and evapo-
rating under reduced pressure. Purification was carried out on
silica using 7:3 cyclohexane: EtOAc as an eluent. Product was
recovered as an off-white solid (5.32 g, 13.9 mmol, 69 %
yield).
1H NMR (500 MHz, Chloroform-d)
d 7.18 (d,
J = 8.7 Hz, 4H, ArH), 6.86 (d, J = 8.7 Hz, 4H, ArH),
5.89 (q, J = 7.1 Hz, 2H ArCHN), 3.85–3.78 (m, 8H,
overlap of C=OCH2N and OCH3), 3.50 (d, J = 16.4 Hz,
2H C=OCH2N), 1.51 (d, J = 7.1 Hz, 6H, CH3). 13C NMR
(126 MHz, Chloroform-d) d 163.9 (C=O), 159.5 (ArOMe),
130.4 (Ar), 128.8 (Ar), 114.3 (Ar), 55.5 (CHCH3), 49.9
(OCH3), 44.7 (NCH2C=O), 15.5 (CHCH3). HR-MS: m/z
Calcd for C22H26N2O4Na (M ? Na)?: 405.1790; found:
405.1786.
(3R,6S)-5-Ethoxy-6-isopropyl-1-(4-methoxybenzyl)-3-
((4-oxocyclohexyl)methyl)-1,6-dihydropyrazin-2(3H)-
one (22)
1H NMR (300 MHz, Chloroform-d)
d 7.13 (d,
J = 8.2 Hz, 2H, ArH), 6.85 (d, J = 8.1 Hz, 2H, ArH), 5.38
(d, J = 14.7 Hz, 1H, ArCHHN), 4.19–3.99 (m, 4H), 3.92–
3.75 (m, 4H), 3.73–3.50 (m, 1H, CHCH(CH3)2), 2.47–1.94
(m, 8H), 1.85–1.68 (m, 1H, NCH(CH2CH)), 1.53–1.35
(m, 2H), 1.26 (m, 4H), 1.05 (d, J = 6.9 Hz, 3H,
CH(CH3)(CH3)), 0.92 (d, J = 6.6 Hz, 3H, CH(CH3)(CH3)).
13C NMR (126 MHz, Chloroform-d) d 212.0 (C=O), 171.8
(O=CN), 159.5 (OC=N), 156.3 (ArOMe), 130.5 (Ar), 128.2
(Ar), 114.2 (Ar). 61.5 (CHCH(CH3)2), 61.0 (O=CCHN=C),
55.4 (OCH3), 50.7 (OCH2CH3), 46.3 (CH2Ar), 40.5
(CH2C=OCH2), 40.3 (CH2C=OCH2), 34.0 (CH2CH2C=O),
33.2 (CH2CH2C=O), 32.0 (CH(CH3)2) 31.7 (O=CCH
(CH2CHC5H8O), 20.1 (CH3)CH(CH3)), 17.6 (CH3)CH
(CH3)), 14.4 (OCH2CH3). HR-MS: m/z Calcd for
C24H34N2O4Na (M ? Na)?: 437.2416; found: 437.2417.
18 (1 g, 3.3 mmol) was placed in an oven-dried two-
necked flask and dissolved in dry THF (20 mL). The
solution was cooled to 0 °C and 4.29 mL of 1 M LHMDS
solution in THF were added slowly. The reaction was
stirred for 30 min during which time the solution devel-
oped a noticeably brighter orange colour. The solution was
cooled to -78 °C with a liquid nitrogen/EtOAc bath and a
solution of 12 (1.17 g, 3.3 mmol) in dry THF was added
dropwise. The mixture was stirred for 2 h, then the cold
bath was removed and the mixture was allowed to reach
room temperature. The reaction was worked up by pouring
the reaction mixture into a mixture of deionised water
(100 mL) and EtOAc (100 mL). The layers were separated
and the aqueous layer was extracted two more time with
20
½aꢁD ¼ ꢂ26:4ꢃðc ¼ 0:99; CHCl3Þ.
123

The synthesis and biological testing
1165
1H NMR (500 MHz, Chloroform-d) d 7.11 (m, 4H,
ArH), 6.88 (d, J = 8.5 Hz, 4H, ArH), 5.77 (dq, J = 30.7,
7.1 Hz, 2H, CH3CH), 3.88 (d, J = 17.1 Hz, 1H,
C=OCHHN), 3.82 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.75
(dd, J = 11.9, 3.7 Hz, 1H, C=OCH(C7H11O)N), 3.63 (d,
J = 17.0 Hz, 1H, C=OCHHN), 2.36 (dd, J = 10.0, 4.7 Hz,
4H, CH2C=OCH2), 2.33–2.27 (m, 1H), 1.96 (m, 2H,
CHHCH2C=OCH2CHH), 1.83 (m, 1H, CHH(C6H9O)),
1.63 (m, CHH(C6H9O)), 1.56 (d, J = 7.1 Hz, 3H,
CH3CH), 1.48 (d, J = 7.1 Hz, 3H, CH3CH), 1.45–1.31 (m,
2H, CHHCH2C=OCH2CHH). 13C NMR (126 MHz, Chlo-
roform-d) d 211.2 (C=O), 166.7 (O=CN), 165.4 (O=CN),
159.5 (ArOMe), 159.5 (ArOMe), 131.2 (CHAr), 130.9
(CHAr), 128.4 (ArH), 128.3 (ArH), 114.4 (ArH), 114.3
(ArH), 55.5 (OCH3), 55.4 (OCH3), 55.3 (NCHC=O), 51.5
(CHCH3), 49.7 (CHCH3), 44.6 (NCH2C=O), 40.8
(CH2C=OCH2), 40.5 (CH2C=OCH2), 39.9 (CH2(C6H9O)),
34.0 (CH2CH2C=O), 33.0 (CH2(CHC5H8O)), 31.8
(CH2CH2C=O), 17.7 (CHCH3), 15.9 (CHCH3). HR-MS:
m/z Calcd for: C29–H36N2O5Na (M ? Na)?: 515.2522;
1,4-Bis((S)-1-(4-methoxyphenyl)ethyl)-3-((4-
oxocyclohexyl)methyl)piperazine-2,5-dione (25)
20 (1.29 g, 3.39 mmol) was placed in an oven-dried two-
necked flask and dissolved in dry THF (12.5 mL) under
nitrogen. The solution was cooled to 0 °C and 4.1 mL of
1 M LHMDS solution in THF were added slowly. The
reaction was stirred for 30 min during which time the
solution developed a noticeably brighter orange colour.
The solution was cooled to -78 °C with a liquid nitrogen/
EtOAc bath and a solution of 12 (1.2 g, 3.39 mmol) in dry
THF (10 mL) was added dropwise. The mixture was stirred
for 10 h then the cold bath was removed and the mixture
was warmed to room temperature. The reaction was
worked up by pouring the reaction mixture into a mixture
of deionised water (50 mL) and EtOAc (50 mL). The
layers were separated and the aqueous layer was extracted
two more time with EtOAc (40 mL). The combined
organic layer was washed with brine (40 mL), dried over
Na2SO4 and the solvent was removed under vacuum to
give crude 24 as a yellow oil; 1.32 g (2.06 mmol) of this
was dissolved in a mixture of acetic acid, THF and
deionised water (16:4:4 mL). The mixture was stirred for
10 h at room temperature when TLC showed consumption
of starting material. The solvent was removed under high
vacuum and the residue was dissolved in a mixture of
deionised water (50 mL) and EtOAc (50 mL). The layers
were separated and the aqueous layer was extracted two
more times with EtOAc (40 mL). The combined organic
layer was washed with brine (40 mL), dried over Na2SO4
and the solvent was removed under vacuum to give the
crude alcohol as a yellow oil. Oxalyl chloride (0.21 mL,
2.4 mmol) was dissolved in dry CH2Cl2 (7 mL) and cooled
to -78 °C using an ethyl acetate/liquid nitrogen bath. To
the stirred solution, DMSO (0.38 mL, 5.3 mmol) was
added slowly. The mixture was stirred for a further 15 min
and the dried crude product (0.87 g, 2 mmol) was added
dropwise as a solution in CH2Cl2 (5 mL). The mixture was
stirred for a further hour maintaining the temperature at
-78 °C. Triethylamine (1.3 mL, 9.62 mmol) was added
and the solution was allowed to warm to -10 °C. The
mixture was stirred for a further 4 h and the colour had
darkened from yellow to brown. The mixture was diluted
with CH2Cl2, filtered to remove sieves and washed with
saturated ammonium chloride solution, water and brine.
The organic phase was dried with MgSO4 and the solvent
was removed under reduced pressure. The resulting brown
residue was purified on silica using 3:2 EtOAc:cyclohexane
as an eluent to give the product in a diastereomeric ratio of
0.81–0.19. Combined overall yield for both isomers was
651 mg (39 %). The analysis for the major isomer
(527 mg) is reported here.
20
found 515.2523. ½aꢁD ¼ ꢂ214:2ꢃðc ¼ 1:12; CHCl3Þ.
(3R,6S)-5-Ethoxy-6-isopropyl-1-(4-methoxybenzyl)-3-
((4-oxocyclohex-2-enyl)methyl)-1,6-dihydropyrazin-
2(3H)-one (23)
A 1 M solution of iodic acid in DMSO was made up and
heated at 80 °C in the absence of light for 1 h; 0.36 mL of
this solution was added to a solution of 22 (75 mg,
0.18 mmol) in DMSO (0.2 mL) and cyclohexene (0.1 mL).
The mixture was heated at 50 °C for 8 h in the absence of
light, when TLC showed consumption of starting material.
Work-up was performed by pouring the mixture into a
mixture of EtOAc (25 mL) and deionised water (25 mL).
The layers were separated and the aqueous layer was
extracted with EtOAc (20 mL) a further five times. The
combined organic layer was washed with brine (25 mL)
dried over Na2SO4 and the solvent was removed under
vacuum. The crude product was purified on silica using a
95:5 mixture of EtOAc:MeOH as an eluent, recovering
50 mg of product as a yellow oil (67 % crude yield). NMR
data showed that the product was contaminated with
*15 % unknown contaminant that could not be separated.
The new stereocentre at the c carbon was a 50:50 mixture
of R and S, the diastereomers could not be separated by
silica chromatography and the 1H NMR reported here
refers to the mixture of diastereomers.
1H NMR (300 MHz, Chloroform-d) d 7.21–7.09 (m, 2H,
ArH), 6.91–6.80 (m, 3H), 6.02–5.94 (m, 1H, O=CCH=CH),
5.41–5.35 (m, 1H, ArCHHN), 4.23–3.99 (m, 4H), 3.95–3.56
(m, 4H), 2.86–2.78 (m, 2H, O=CCH2CH2), 2.48–1.93 (m,
3H), 1.84–1.67 (m, 1H, NCH(CH2CH)), 1.53–1.35 (m, 2H),
123

1166
K. Robertson et al.
1.31–1.20 (m, 3H), 1.10–1.01 (m, J = 3H, CH(CH3)(CH3)),
0.97–0.93 (m, 3H, CH(CH3)(CH3)). 13C NMR (126 MHz,
Chloroform-d), diastereomer 1: d 198.9 (C=O), 173.5
(O=CN), 159.6 (OC=N), 156.3 (ArOMe), 142.6 (O=
CCH=CH), 129.7 (Ar), 128.7 (Ar), 124.7 (O=CCH=CH),
115.9 (Ar). 62.3 (CHCH(CH3)2), 61.1 (O=CCHN=C), 55.4
(OCH3), 50.8 (OCH2CH3), 46.5 (CH2Ar), 40.8 (CH2C=
OCH=CH), 36.7 (CH2(C6H7O)), 33.8 (CH(C5H6O)), 30.3
(CH2CH2C=O), 27,1 (CH(CH3)2), 20.0 (CH3)CH(CH3)),
18.0 (CH3)CH(CH3)), 14.0 (OCH2CH3); diastereomer 2: d
198.8 (C=O), 173.4 (O=CN), 159.6 (OC=N), 156.3
(ArOMe), 139.4 (O=CCH=CH), 129.6 (Ar), 128.7 (Ar),
123.9 (O=CCH=CH), 114.2 (Ar), 61.6 (CHCH(CH3)2), 60.9
(O=CCHN=C), 55.4 (OCH3), 50.8 (OCH2CH3), 46.4
(CH2Ar), 40.8 (CH2C=OCH=CH), 36.6 (CH2(C6H7O)),
33.8 (CH(C5H6O)), 30.3 (CH2CH2C=O), 27.1 (CH(CH3)2),
20.0 ((CH3)CH(CH3)), 18.0 (CH3)CH(CH3)), 14.0
(OCH2CH3). HR-MS: m/z Calcd for C24H32N2O4Na
(M ? Na)?: 437.2261; found: 437.2254.
overlap), 3.87 (d, J = 17.2 Hz, 1H), 3.82 (s, 3H, overlap),
3.81 (s, 3H, overlap), 3.79–3.74 (m, 1H, overlap), 3.63 (d,
J = 17.2 Hz, 1H), 2.52–2.34 (m, 2H, overlap), 2.12–1.64
(m, 5H, overlap), 1.56 (d, J = 7.2 Hz, 3H, overlap), 1.41
(d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, Chloroform-d),
diastereomer 1: d 199.2 (C=O), 166.2 (NC=O), 165.3
(NC=O), 159.6 (ArOMe), 159.5 (ArOMe), 153.2 (CH=
CHC=O), 130.9 (Ar), 130.6 (Ar), 129.9 (CH=CHC=O),
128.4 (Ar), 128.3 (Ar), 114.5 (Ar), 114.3 (Ar), 55.4
(OCH3), 55.4 (OCH3), 55.1 (NCHC=O), 51.8 (CCH3), 51.6
(CCH3), 44.5 (NCH2C=O), 38.6 (NCHCH2(C6H7O)), 36.7
(CH2CH2C=O), 33.2 (DKPCH2CH), 29.8 (C=OCH2CH2),
17.8 (CHCH3), 15.9 (CHCH3); diastereomer 2: d 198.9
(C=O), 166.1 (NC=O), 165.2 (NC=O), 159.5 (ArOMe),
159.5 (ArOMe), 151.7 (CH=CHC=O), 129.7 (CH=
CHC=O), 130.8 (Ar), 130.6 (Ar), 128.3 (Ar), 128.3 (Ar),
114.4 (Ar), 114.3(Ar), 55.4 (OCH3), 55.4 (OCH3), 54.8
(NCHC=O), 49.8 (CCH3), 49.7 (CCH3), 44.5 (NCH2C=O),
38.6 (NCHCH2(C6H7O)), 36.4 (CH2CH2C=O), 32.5
(DKPCH2CH), 29.8 (C=OCH2CH2), 17.7 (CHCH3), 14.2
(CHCH3). HR-MS: m/z Calcd for: C29–H34N2O5Na
(M ? Na)?: 513.2365; found 513.2359.
(S)-1,4-Bis((S)-1-(4-methoxyphenyl)ethyl)-3-((4-
oxocyclohex-2-enyl)methyl) piperazine-2,5-dione (26)
A 1 M solution of iodic acid in DMSO was made up and
heated at 80 °C in the absence of light for 1 h (caution:
explosion hazard); 0.4 mL of this solution was added to a
solution of 25 (100 mg, 0.2 mmol) in DMSO (0.25 mL)
and cyclohexene (0.15 mL). The mixture was heated at
50 °C for 7 h in the absence of light, when TLC showed
consumption of starting material. Work-up was performed
by pouring the mixture into a mixture of EtOAc (25 mL)
and deionised water (25 mL). The layers were separated
and the aqueous layer was extracted with EtOAc (20 mL) a
further five times. The combined organic layer was washed
with brine (25 mL) dried over Na2SO4 and the solvent was
removed under vacuum. The crude product was purified on
silica using a 95:5 mixture of EtOAc:MeOH as an eluent.
Product (79 mg) was recovered as a pale yellow oil. NMR
data showed that the product was contaminated with
*10 % unknown contaminant that could not be separated.
The new stereocentre at the c carbon was a 50:50 mixture
of R and S; the diastereomers could not be separated by
silica gel chromatography.
1H NMR (400 MHz, Chloroform-d), diastereomer 1: d
7.15–7.06 (m, 4H, overlap), 6.93 (dd, J = 10.6, 3.5 Hz,
1H), 6.90–6.86 (m, 4H, overlap), 6.00–5.97 (m, 1H),
5.85–5.68 (m, 2H), 3.89 (d, J = 17.2 Hz, 1H), 3.82 (s, 3H),
3.81 (s, 3H, overlap), 3.79–3.74 (m, 1H, overlap), 3.63 (d,
J = 17.2 Hz, 1H, overlap), 2.52–2.34 (m, 2H, overlap),
2.15–1.64 (m, 5H, overlap), 1.56 (d, J = 7.2 Hz, 3H), 1.46
(d, J = 7.1 Hz, 3H); diastereomer 2: d 7.15–7.06 (m, 4H,
overlap), 6.90–6.86 (m, 4H), 6.68 (dd, J = 10.1, 2.6 Hz,
1H), 6.02 (broad d, J = 10.3 Hz, 1H), 5.85–5.68 (m, 2H,
S-3-((4-Oxocyclohexyl)methyl)piperazine-2,5-dione
(4)
25 (70 mg, 0.14 mmol) was dissolved in acetonitrile/water
(3 mL: 2 mL), and cerium ammonium nitrate was added
(311 mg, 0.57 mmol). The mixture was stirred at room
temperature until TLC showed consumption of starting
material (4 h). The reaction mixture was purified directly
on silica using 4:1 CH2Cl2:MeOH as an eluent. The
product (20.8 mg, 66 % yield) was recovered as a white
solid.
1H NMR (500 MHz, deuterium oxide) d 4.25–4.18 (m,
1H, C=OCH(CH2C6H9O), overlaps with peak at 4.22), 4.22
(d J = 18.0 Hz, 1H, C=OCHHNH, overlaps with multiplet
at 4.25–4.18) 4.06 (d, J = 18.2 Hz, 1H, C=OCHHNH),
2.63–2.50 (m, 2H, CHHC=OCHH), 2.51–2.39 (m, 2H,
CHHC=OCHH), 2.22–1.85 (m, 5H), 1.65–1.50 (m, 2H).
13C NMR (126 MHz, deuterium oxide) d 219.9 (C=O),
171.5 (HNC=O), 169.2 (HNC=O), 53.6 (HNCHC=O), 44.3
(HNCH2C=O), 40.6 (CH2C=OCH2), 40.4 (CH2C=OCH2),
39.1 (CH2CH(C5H8O), 32.9, 32.0, 31.6. HR-MS: m/z Calcd
for: C11H14N2O3Na (M ? Na)?: 247.1061; found
247.1059.
(3S)-3-((4-Oxocyclohex-2-enyl)methyl)piperazine-2,
5-dione (5)
26 (72 mg, 0.15 mmol) was dissolved in acetonitrile/water
(2 mL: 0.5 mL) and cerium ammonium nitrate was added
(321 mg, 0.59 mmol). The mixture was stirred at room
123

The synthesis and biological testing
1167
temperature until TLC showed consumption of starting
material (5 h). The solvent was removed under high vac-
uum and the residue was purified directly on silica using
9:1 CH2Cl2:MeOH as an eluent. The product (9.4 mg,
28 % yield) was recovered as an off-white solid; the dia-
stereomers could not be separated.
13C NMR (126 MHz, deuterium oxide)
d
211.5
(CH2C=OCH2), 178.1 (HOC=O), 177.0 (HNC=O), 60.0
(CHisoPr), 52.5 (H2NCHC=O), 38.3 (CH2C=OCH2), 37.8
(CH2C=OCH2), 36.3 (CHCH2C6H9O), 32.2 (CH3CHCH3),
30.6 (CH2CH2C=OCH2CH2), 29.9 (CH2CHC5H8O), 18.2
(CH3CHCH3), 17.5 (CH3CHCH3). HR-MS: m/z Calcd for
C14H25N2O4: 285.1815; found: 285.1821.
1H NMR (500 MHz, deuterium oxide), diastereomer 1: d
7.15–7.06 (m, 1H, O=CCH=CH), 6.10–6.02 (m, 1H,
O=CCH=CH), 4.27–4.15 (m, 2H), 4.10–4.01 (m, 1H,
C=OCHHNH), 2.71–2.35 (m, 3H), 2.23–2.15 (m, 1H,
CHHCH2C=O), 1.99–1.90 (m, 2H, CH2C6H7O), 1.79–1.72
(m, 1H, CHHCH2C=O); diastereomer 2: d 7.08–7.00 (m, 1H,
O=CCH=CH), 6.05–5.99 (m, 1H, O=CCH=CH), 4.27–4.15
(m, 2H, overlap), 4.04–3.98 (m, 1H, C=OCHHNH),
2.71–2.35 (m, 3H, overlap), 2.23–2.15 (m, 1H,
CHHCH2C=O, overlap), 1.93–1.88 (m, 2H, CH2C6H7O,
overlap), 1.75–1.69 (m, 1H, CHHCH2C=O, overlap). 13C
NMR (126 MHz, deuterium oxide), diastereomer 1: d 199.3
(O=C), 171.0 (O=CN), 169.1 (O=CN), 153.5 (O=CCH=
CH), 130.0 (O=CCH=CH), 53.5 (O=CHN), 43.7 (O=CH2N),
41.0 ((NCHCH2(C6H7O)), 36.9 (O=CCH2CH2), 32.5
(CH2CHC5H6O), 28.5 (O=CCH2CH2); diastereomer 2: d
199.0 (O=C), 170.9 (O=CN), 168.7 (O=CN), 153.4,
(O=CCH=CH), 130.0 (O=CCH=CH), 53.3 (O=CHN), 43.6
(O=CH2N), 41.0 ((NCHCH2(C6H7O)), 36.8 (O=CCH2CH2),
32.5 (CH2CHC5H6O), 28.4 (O=CCH2CH2). HR-MS: m/z
Calcd for: C11H14N2O3Na (M ? Na)?: 245.0902; found
245.0910.
(S)-2-((R)-2-Amino-3-(4-oxocyclohex-2-
enyl)propanamido)-3-methylbutanoic acid (7)
23 (79 mg, 0.19 mmol) was dissolved in a mixture of
acetonitrile and deionised water (2:0.5 mL) cerium (IV)
ammonium nitrate (208 mg, 0.38 mmol) was added and the
mixture was stirred at room temperature until TLC showed
consumption of starting material (4 h). The solvent was
removed under high vacuum and the residue was partially
purified using silica with 10 % MeOH in CH2Cl2 as an
eluent. The residue was dissolved in 1 M hydrochloric acid
(2.5 mL) and the mixture was stirred at 45 °C for 9 h. The
solvent was removed under high vacuum and dissolved in
water (10 mL) and EtOAc (10 mL). The layers were sep-
arated and the aqueous layer washed with EtOAc (5 mL)
two more times. The product as the hydrochloride salt was
recovered as an off-white solid after drying the aqueous
layer under high vacuum. (11.5 mg, 19 % yield). The
mixture of diastereomers could not be separated and the 1H
NMR reported here refers to the mixture.
1H NMR (500 MHz, Deuterium oxide) d: 6.99–6.93 (m,
1H, O=CCH=CH), 6.07–6.03 (m, 1H, O=CCH=CH),
(S)-2-((R)-2-Amino-3-(4-oxocyclohexyl)propanamido)-
3-methylbutanoic acid (6)
4.42–4.33 (m,
1 H, NH2CH), 4.09–3.99 (m, 1H,
CHCH(CH3)2), 2.78–2.62 (m, 3H), 2.36–2.25 (m, 2H,
CHCH2(C6H9)), 2.07–1.85 (m, 5H), 1.05–1.00 (m, 3H,
(CH3)CH(CH3)), 0.96–0.89 (m, 3H, (CH3)CH(CH3)). 13C
NMR (126 MHz, D2O), diastereomer 1: d: 198.9
(CH2C=OCH2), 178.1 (HOC=O), 177.0 (HNC=O), 151.5
(O=CCH=CH), 123.7 (O=CCH=CH), 60.0 (CHisoPr), 52.7
(H2NCHC=O), 38.3 (CH2CH2C=O), 36.7 (CHCH2C6H9O),
31.4 (CH3CHCH3), 30.6 (CH2CH2C=O), 29.9 (CH2CH
C5H8O), 17.5 (CH3CHCH3), 15.3 (CH3CHCH3); diaste-
reomer 2: d 198.8 (CH2C=OCH2), 178.1 (HOC=O),
176.5.0 (HNC=O), 151.3 (O=CCH=CH), 123.4
(O=CCH=CH), 59.8 (CHisoPr), 52.5 (H2NCHC=O), 38.1
(CH2C=OCH2), 36.3 (CHCH2C6H9O), 31.2 (CH3CHCH3),
30.3 (CH2CH2C=O), 29.9 (CH2CHC5H8O), 18.1
(CH3CHCH3), 17.5 (CH3CHCH3). HR-MS: m/z Calcd for
C14H23N2O4: 285.1658; found: 285.1652.
22 (120 mg, 0.29 mmol) was dissolved in a mixture of
acetonitrile and deionised water (4:1 mL) cerium (IV)
ammonium nitrate (323 mg, 0.59 mmol) was added and the
mixture was stirred at room temperature until TLC showed
consumption of starting material (3.5 h). The solvent was
removed under high vacuum and the residue was partially
purified using silica with 10 % MeOH in CH2Cl2 as an
eluent. The residue was dissolved in 1 M hydrochloric acid
(3 mL) and the mixture was stirred at 45 °C for 9 h
monitoring by TLC. The solvent was removed under high
vacuum and dissolved in water (10 mL) and EtOAc
(10 mL). The layers were separated and the aqueous layer
was washed with EtOAc (5 mL) two more times. The
product as the hydrochloride salt was recovered as an off-
white solid after drying the aqueous layer under high
vacuum. (40 mg, 43 % yield).
1H NMR (500 MHz, deuterium oxide) d 4.40–4.32 (m, 1
H, NH2CH), 4.02 (d, J = 7.5 Hz, 1H, CHCH(CH3)2),
2.79–2.61 (m, 5H), 2.36–2.25 (m, 2H, CHCH2(C6H9)),
2.09–1.83 (m, 5H), 1.03 (d, J = 7.1 Hz, 3H,
(CH3)CH(CH3)), 0.91 (d, J = 6.9 Hz, 3H, (CH3)CH(CH3)).
Antibacterial testing
Non-resistant strains of S. aureus, E. coli, S. cerevisiae and
C. albicans (all obtained from the Microbiology Culture
Collection, UCD School of Biomolecular and Biomedical
123

1168
K. Robertson et al.
synthesis of quaternary alpha-amino acids from diketopiperazine
templates. Org Biomol Chem 5:2138–2147
Science) were employed to test compounds 4–7 via the disc
diffusion assay. The compounds were prepared in sterile
water (20 mg mL-1) and 25 lL was added to sterile discs,
which were placed onto inoculated agar plates.
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Jedrzejczak R, Wojciechowski M, Andruszkiewicz R, Sowinski P,
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Antibiotic-resistant strains of E. coli DSM1103, methi-
cillin resistant S. aureus ATCC 43300 plus in-house clin-
ical isolates of E. coli and MRSA were also used to assess
the antibacterial activity of 4–7. Serial dilutions of the test
compounds were prepared in Mueller–Hinton broth (MHB)
and aliquots (100 lL) transferred to wells of a microtitre
plate. Bacterial cultures were incubated at 37 °C overnight,
diluted 9 1,000 in phosphate buffered saline and 5 lL of
this was used to inoculate wells containing the test com-
pounds. The plate was then incubated at 37 °C for 18 h
using an OminologÒ automated incubator (Biolog Inc. CA
94545, USA).
Acknowledgments This projected was funded under the Irish
Government’s Programme for Research Training in Third Level
Institutes (PRTLI). The authors thank Dr Maria Martins of the UCD
Centre for Food Safety for conducting the microtitre plate assays.
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and selective alternative reagents to IBX for the dehydrogenation
of aldehydes and ketones. Angewandte Chemie-Int Edition
41(8):1386–1389
Conflict of interest The authors declare that they have no conflict
of interest.
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diketopiperazine synthesis using phase transfer catalysis. Tetra-
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of uncommon tripeptides derived from 2,6-diaminopimelic acid
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