A comparative study of antimicrobial properties of cyclo(L-Pro- L-Asp) with its 2-ketopiperazine analog

Article abstract of DOI:10.1007/s00044-013-0836-5

Cyclo(L-Pro- L-Asp) diketopiperazine was synthesized and its antimicrobial properties were compared against its 2-ketopiperazine analog. The results indicate the significance of the peptide bond in antimicrobial property of cyclo(L-Pro- L-Asp) against various pathogenic bacteria and fungi with potent inhibitory effect over the 2-ketopiperazine analog. Cyclo(L-Pro- L-Asp) exhibited a higher inhibitory activity against Penicillium expansum (MIC 8 μg/mL) than the standard fungicide amphotericin B (MIC 64 μg/mL). The most potent activity by cyclo(L-Pro- L-Asp) was exhibited against Trichophyton rubrum (MIC 2 μg/mL). Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0836-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2377–2385
DOI 10.1007/s00044-013-0836-5
ORIGINAL RESEARCH
A comparative study of antimicrobial properties
of cyclo(L-Pro- L-Asp) with its 2-ketopiperazine analog
•
Chandrasekhar Challa Nishanth Kumar
•
•
Manju John Ravi S. Lankalapalli
Received: 12 July 2013 / Accepted: 7 October 2013 / Published online: 17 October 2013
Ó Springer Science+Business Media New York 2013
Abstract Cyclo(L-Pro- L-Asp) diketopiperazine was syn-
thesized and its antimicrobial properties were compared
against its 2-ketopiperazine analog. The results indicate the
significance of the peptide bond in antimicrobial property
of cyclo(L-Pro- L-Asp) against various pathogenic bacteria
and fungi with potent inhibitory effect over the 2-keto-
piperazine analog. Cyclo(L-Pro- L-Asp) exhibited a higher
inhibitory activity against Penicillium expansum (MIC
8 lg/mL) than the standard fungicide amphotericin B
(MIC 64 lg/mL). The most potent activity by cyclo(L-Pro-
L-Asp) was exhibited against Trichophyton rubrum (MIC
2 lg/mL).
antitumor, antiviral, antihyperglycaemic, and antimicrobial
effects (Martins and Carvalho, 2007; Ortiz-Castro et al.,
2011; Chen et al., 2012). The antimicrobial activities of
DKPs are well studied, that led to culmination of these
privileged structures find application in pharmaceutical
industry as prospective antibiotics (de Carvalho and Abra-
ham, 2012). Recently, we reported six proline residues
containing cyclodipeptides cyclo(D-Pro- L-Leu), cyclo
(L-Pro- L-Met), cyclo(D-Pro- L-Phe), cyclo(L-Pro- L-Phe), cyclo
(L-Pro- L-Tyr), and cyclo(L-Pro- D-Tyr) from ethyl acetate
extract of a Bacillus strain associated with a new rhabditid
entomopathogenic nematode (Kumar et al., 2013). These
cyclic dipeptides exhibited potent inhibitory activity against
both medicinally and agriculturally important bacteria and
fungi. Reports from literature suggest that proline-based
DKPs pertaining to L-amino acids with neutral, acidic, and
basic side chains were very less studied for their antimicro-
bial properties (Li et al., 2012, 2013). On the contrary,
antimicrobial effects of other proteinogenic amino acids
with aliphatic and aromatic side chains were extensively
studied (Graz et al., 1999, 2001; Jiang et al., 2000; Yang
et al., 2002; Rhee, 2003; Bitzer et al., 2006; Lind et al., 2007;
Ben Ameur Mehdi et al., 2009; Klausmeyer et al., 2009; Qi
et al., 2009; Ryan et al., 2009; Kumar et al., 2012; Park et al.,
2012; Wang et al., 2012; Mehnaz et al., 2013; Tian et al.,
2013). Therefore, we have synthesized herewith cyclo(L-
Pro- L-Asp) and evaluated its antimicrobial properties
against different human and agriculturally important bacte-
ria and fungi. In order to study the effect of a peptide bond in
antimicrobial activity we have also synthesized a 2-keto-
piperazine analog of cyclo(L-Pro- L-Asp) which exhibited a
diminished antimicrobial activity. The present work is the
first report for evaluation of antimicrobial property of
cyclo(L-Pro- L-Asp), which demonstrates an excellent anti-
fungal activity than the standard fungicide amphotericin B
Keywords Diketopiperazines Á 2-Ketopiperazine Á
Minimum inhibitory concentration Á Disk diffusion assay Á
Antimicrobial activity
Introduction
2,5-Diketopiperazines (DKPs) are cyclodipeptides which
have recently gained prominence in medicinal chemistry
owing to their small yet rigid peptide backbone with non-
planar structure endowed with sufficient diversity elements
around its periphery, and due to its presence as subunits
embedded in numerous natural products (Huang et al.,
2010; Borthwick, 2012). DKPs are widespread in nature
exhibiting a broad gamut of biological properties such as
C. Challa Á N. Kumar Á M. John Á R. S. Lankalapalli (&)
Agroprocessing and Natural Products Division, National
Institute for Interdisciplinary Science & Technology (NIIST),
Council of Scientific and Industrial Research (CSIR),
Thiruvananthapuram 695019, Kerala, India
e-mail: ravishankar@niist.res.in
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Med Chem Res (2014) 23:2377–2385
Scheme 1 Synthesis of DKP 3
and we also showed the significance of the peptide bond for
antimicrobial activity of diketopiperazine.
Chemistry
Scheme 2 Synthesis of 2-ketopiperazine analog 6
Synthesis of DKP 3 (Scheme 1) started by coupling
between N-Boc-L-aspartic acid 4-benzyl ester and L-proline
methyl ester in the presence of EDCI and HOBt to afford
dipeptide 1 in a good yield. Boc group removal in the
presence of neat formic acid followed by ring closure under
reflux in toluene afforded DKP 2 in 80 % yield (two steps),
Finally, Boc group deprotection in the presence of TFA and
debenzylation using Pd/C (10 %) in the presence of H2
afforded 2-ketopiperazine analog 6 in a quantitative yield,
1
as confirmed by H NMR indicating loss of benzylic and
t-butyl proton signals.
1
by following a reported procedure (Crick et al., 2011). H
NMR of DKP 2 shows a loss of singlet (3H) corresponding
to OCH3 of methyl ester and a loss of t-butyl protons
confirming cyclization of compound 1. Final debenzylation
using Pd/C (10 %) in the presence of H2 afforded DKP 3,
as indicated in the 1H NMR showing disappearance of
down-field benzylic protons at d 5.03 and d 7.23 of DKP 2
and appearance of a singlet corresponding to COOH at d
7.23 in DKP 3. 2-Ketopiperazine analog 6 (Scheme 2) was
synthesized in five steps from (S)-(?)-prolinol. Coupling of
(S)-prolinol with N-Boc-L-aspartic acid 4-benzyl ester in
the presence of BOP and HOBt afforded dipeptide 4.
Mesylation of 1° alcohol followed by cyclization in the
presence of Cs2CO3 afforded the cyclized product 5 at
63 % yield (two steps). An apparent change in multiplicity
of H-9 (m) in dipeptide 4 to doublet in H-2 corresponding
to 2-ketopiperazine 5 of the trans-coupling proton with
H-3, as expected, indicates cyclized product formation.
Biological results
Test microorganisms
DKP 3 and 2-ketopiperazine 6 were screened for their
antimicrobial activity against three Gram-positive bacteria
(Bacillus subtilis MTCC 2756, Staphylococcus aureus
MTCC 902, and S. epidermidis MTCC 435), three Gram-
negative bacteria (Escherichia coli MTCC 2622, Klebsiella
pneumoniae MTCC 109, and Pseudomonas aeruginosa
MTCC 2642), as well as eight fungi (Aspergillus flavus
MTCC 183, A. fumigatus MTCC 3376, A. niger MTCC
277, A. tubingensis MTCC 2425, Candida albicans MTCC
3017, C. tropicalis MTCC 184, Penicillium expansum
MTCC 2006, and Trichophyton rubrum MTCC 296). All
the test microorganisms were procured from Microbial
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Med Chem Res (2014) 23:2377–2385
2379
Table 1 Antibacterial properties of peptides 3 and 6
Organism
MIC (lg/mL)
Zone of inhibition (diameter in mm)
2-Ketopiperazine 6 DKP 3
2-Ketopiperazine 6
DKP 3
Ciprofloxacin
Ciprofloxacin
B. subtilis
125
64
32
16
8
2
15
19 1.15
17
0
24
1
30
32
1
0
E. coli
1
26 1.15
K. pneumoniae
P. aeruginosa
S. aureus
32
0.5
1
1
31
26
21
1
0
0
33 1.15
33 0.57
125
250
64
16
64
64
14 0.57
16 1.73
1
31
1
S. epidermidis
0.25
23
1
24 1.73
31 1.15
MIC results are an average of three replications and zone of inhibition were an average of three replications standard deviation
Type Culture Collection and Gene Bank, Institute of
Microbial Technology (IMTECH), CSIR, Chandigarh,
India.
concentrations which is considerably higher for 2-keto-
piperazine 6.
Antifungal activity
Antibacterial activity
The MIC values of the test compounds against test fungi
were determined by microbroth dilution method as per the
guidelines of Clinical and Laboratory Standard Institute
(formerly, the National Committee for Clinical Laboratory
Standards) (CLSI, 2008, 2012c), with RPMI 1640 medium
containing L-glutamine, without sodium bicarbonate and
buffered to pH 7.0. Two-fold serial dilutions of the peptide
compounds were prepared in media for an amount of
100 lL per well in 96-well U-bottom microtiter plates
(Tarson, Mumbai, India). The test fungal suspensions were
further diluted in media and a 100 lL volume of these
diluted inoculums was added to each well of the plate,
resulting in a final inoculum of 0.5 9 104–2.5 9 104 CFU/
mL for Candida spp. and 0.4 9 104–5 9 104 CFU/mL for
other fungi. The final concentration of peptide compounds
ranging from 1 to 1000 lg/mL and amphotericin B served
as the standard drug control. The microtiter plates were
incubated at 37 °C for 48 h for Candida species and 30 °C
for 72 h for other fungi. MIC values recorded in Table 2
confirm a similar trend as antibacterial activity with DKP 3
showing more inhibition than 2-ketopiperazine 6 against all
test microorganisms. DKP 3 exhibited profound inhibitory
effect against A. fumigates, A. tubingensis, C. albicans, C.
tropicalis, and P. expansum over 2-ketopiperazine 6.
Interestingly, DKP 3 was twice more effective than stan-
dard drug amphotericin B in inhibition against the majority
of the test fungal species with a significant one being
against P. expansum (MIC 8 lg/mL) over amphotericin B
(MIC 64 lg/mL). Against A. fumigatus DKP 3 (MIC
32 lg/mL) was less inhibitory than amphotericin B (MIC
16 lg/mL). Both DKP 3 and the drug exhibited equipo-
tency (MIC 16 lg/mL) against A. flavus. The test com-
pounds were also screened for their antifungal activity
against test fungi by disk diffusion method (CLSI, 2009,
2010). The fungal cultures were grown on potato dextrose
The minimum inhibitory concentration (MIC, expressed in
lg/mL) for bacteria was determined according to the
method described by Clinical and Laboratory Standards
Institute (CLSI, 2012a), and the MIC values of DKP 3 and
2-ketopiperazine 6 against the test bacteria are listed in
Table 1 along with reference antibiotic ciprofloxacin. Two-
fold serial dilutions of the antibiotic and peptide com-
pounds were made in Mueller–Hinton broth (MHB) to give
concentrations ranging from 1 to 1,000 lg/mL. Test bac-
terial suspension (100 lL) was inoculated in each well to
give a final concentration of 1 9 105 CFU/mL and the
tubes were incubated for 24 h at 37 °C. The growth was
observed both visually and by measuring OD at 600 nm in
triplicate. Ciprofloxacin was used as a positive control and
methanol was used as blank. The MIC values (Table 1)
indicate that with the entire test bacteria, DKP 3 and
2-ketopiperazine 6 were less potent than standard antibiotic
ciprofloxacin. Interestingly, DKP 3 is more potent than
2-ketopiperazine 6 against all bacteria except S. epide-
rmidis which showed equal bioactivity with an MIC value
of 64 lg/mL. Furthermore, the antimicrobial activity was
also determined by the disk diffusion method against test
bacteria (CLSI, 2012b). The test cultures maintained in
nutrient agar slant at 4 °C were subcultured in nutrient
broth to obtain the working cultures approximately con-
taining 1 9 106 CFU/mL. The MIC concentration of the
peptide compounds were incorporated in a 6 mm sterile
disk. Mueller–Hinton (MH) agar plates were swabbed with
each bacterial strain and the test disks were placed along
with the control disks. Ciprofloxacin disks (5 lg/disk) were
used as positive control and plates were incubated over-
night at 37 °C. The zone of inhibition (expressed in mm,
Table 1; Fig. 1) results further bear evidence over stronger
antibacterial potency exhibited by DKP 3 even at MIC
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Med Chem Res (2014) 23:2377–2385
Fig. 1 Antibacterial property of 2-ketopiperazine 6 and DKP 3
Table 2 Antifungal properties of peptides 3 and 6
Organism
MIC (lg/mL)
Zone of inhibition (diameter in mm)
2-Ketopiperazine 6 DKP 3
28
2-Ketopiperazine 6
DKP 3
Amphotericin B
Amphotericin B
22
A. flavus
32
250
64
16
32
16
32
8
16
16
32
64
16
8
24
18
1
0
0
1
A. fumigatus
A. niger
23 1.15
22 1.73
25 1.73
26 1.15
19 1.15
21 1.73
A. tubingensis
C. albicans
C. tropicalis
P. expansum
T. rubrum
125
250
125
500
8
25
26
33
1
0
1
30
31 0.57
24
20 0.57
29
1
15
0
4
14 0.57
13 1.20
0
8
64
4
29 1.20
38
2
28
1
0
1
MIC results are an average of three replications and zone of inhibition were an average of three replications standard deviation
broth (Hi-Media, India) and the mycelial mat of 7-day-old
culture was suspended in normal saline solution and test
inoculum was adjusted to 1–5 9 105 CFU/mL. Inocula
(0.1 mL) were applied on the surface of the potato dextrose
agar plate and spread by using a cotton swab. Subse-
quently, filter paper disks (6 mm in diameter) containing
MIC concentration of test compounds were placed on the
agar plates and incubated at 37 °C for 48 h. The diameter
zone of inhibition measured (in mm, Table 2; Fig. 2)
clearly demonstrated the potent antifungal activity of DKP
3 over 2-ketopiperazine analog 6, similar to disk diffusion
assay results for its antibacterial activity.
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Med Chem Res (2014) 23:2377–2385
2381
Fig. 2 Antifungal property of 2-ketopiperazine 6 and DKP 3
Discussion
pneumoniae, P. aeruginosa, S. aureus, and S. epidermidis.
The fungi investigated are pharmaceutically important
pathogens such as T. rubrum which causes athlete’s foot, A.
fumigatus, C. albicans, C. tropicalis, and organisms
implicated in agriculture such as A. flavus, A. niger, A.
tubingensis, and P. expansum. In order to assess the sig-
nificance of peptide bonds in DKPs we have synthesized
the 2-ketopiperazine analog 6 of DKP 3. The antimicrobial
activity results clearly indicated that 2-ketopiperazine
analog 6 is less effective than DKP 3 against all the test
organisms. Structural deletion of one carbonyl in DKP 3
and its concomitant decrease in antimicrobial effect clearly
reveals, in general, the significance of the peptide bond in
DKPs. Although, the antibacterial activity of DKP 3 was
lesser than standard antibiotic ciprofloxacin against all test
organisms but the antifungal activity of DKP 3 was better
than standard fungicide amphotericin B against most of the
DKPs exhibit a broad-spectrum antimicrobial activity
against different types of test pathogens, but the downside
of this property is its activity-toxicity problem which
remains a key issue to be addressed in clinical applications
(de Carvalho and Abraham, 2012). To date, much attention
was given toward development of drugs for bacteria and
virus but a number of fungal pathogens also pose a serious
threat in infections.
In the introduction we have mentioned that DKPs per-
taining to L-amino acids with acidic and basic side chains
were less investigated for their antimicrobial effects. In
view of this we have synthesized cyclo(L-Pro- L-Asp) (DKP
3) and studied its activity against different bacteria and
fungi. The bacteria tested in the present investigation are
human pathogens such as B. subtilis, E. coli, K.
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Med Chem Res (2014) 23:2377–2385
fungi. It is well known that compounds which function as
antibiotics are not active against fungi and vice versa. The
most potent activity by DKP 3 was exhibited against T.
rubrum (MIC 2 lg/mL) and a higher inhibitory activity
than the standard fungicide amphotericin B (MIC 64 lg/
mL) was shown against P. expansum (MIC 8 lg/mL).
The mechanism of action of various antifungal peptides
involve cell-wall binding, membrane permeabilization,
receptor-mediated internalization, and interaction with
intracellular targets (van der Weerden et al., 2013). The
site of action of Amphotericin B (AmB) involves binding
with ergosterol present in fungal membranes, which results
in the formation of pores leading to leakage of ions as well
as small organic molecules causing osmotic stress and
finally cell death (Brajtburg et al., 1990; Baginski and
Czub, 2009). The key interactions of AmB with ergosterol
involve hydrophobic interaction with steroidal backbone,
pi-interaction between polyene and sterol double bond(s),
and interaction with ionizable polar group such as amine of
sugar moiety. On a comparative analysis, DKP 3 do not
have structural features as of AmB to exhibit the key
interactions with ergosterol. It was shown that the presence
of negative charge on carboxyl group in AmB is not
essential for biological activity (Cheron et al., 1988).
Therefore, the effect of DKP 3 is probably not by binding
to a specific cell-membrane component like ergosterol.
Other common antifungal agents such as azoles induce
fungistasis by the inhibition of cytochrome P-450 lanos-
terol 14a-demethylase responsible for ergosterol biosyn-
thesis (Kelly et al., 1997). The structural feature in azoles
which is also present in human salivary antifungal histatins
(histidine-rich peptides) is an imidazole. It was shown that
replacement of histidine in histatin with alanine resulted in
loss of candidacidal activity, which indicates that histatins
share a common mechanism of action as azole drugs and
thus implying the importance of imidazole side chain (Tsai
and Bobek, 1997). An imidazole side chain at physiologi-
cal pH is uncharged, whereas carboxyl group in DKP 3 is
negatively charged and therefore share no common struc-
tural characteristics. This indicates that DKP 3 antimicro-
bial effect involving binding to 14a-demethylase is
dubious. Finally, the majority of antimicrobial peptides
(AMPs) that exhibit antifungal activities are cationic and
amphipathic in nature which is not the case in DKP 3. The
mode of action of AMPs is characterized by interaction
with fungal membrane causing pore formation and even-
tually leading to membrane degradation (Epand and Vogel,
1999; Soltani et al., 2008). The mechanism of action of
antifungal activity of cyclodipeptides remains largely
unknown. However, Graz et al. (2001) reported the
mechanism of action of cyclodipeptides on receptor
mediation followed by activation of intracellular signaling
and membrane disruption, albeit the actual interaction of
cyclodipeptide with the target may be more complex.
Based on the above findings, the key non-covalent
interactions present in DKP 3 are electrostatic and hydro-
gen bonding which facilitates binding to its microbial tar-
get. In contrast, the 2-ketopiperazine analog 6 is devoid of
one hydrogen bond acceptor which resulted in lessening of
antimicrobial activity than DKP 3. Thus, by comparison of
structural features in DKP 3 and analog 6 it clearly indi-
cates the significance of peptide bond in DKPs for its
antimicrobial property and studies pertaining to the actual
target of DKPs warrant further investigation.
Experimental
All reactions were carried out with distilled solvents under
inert atmosphere. N-Boc-L-aspartic acid 4-benzyl ester,
L-proline methyl ester, and (S)-(?)-prolinol were pur-
chased from Alfa Aesar. Silica gel G-60 F254 aluminum
TLC plates were used to monitor the reactions with short
wavelength ultraviolet light and/or by iodine staining to
visualize the spots. Flash column chromatography was
1
performed on silica gel 230–400 mesh. H and 13C NMR
spectra were recorded at 500 and 125 MHz, respectively.
Chemical shifts are given in ppm using solvent residual
peaks as reference and coupling constants in Hz. HRMS
analysis was recorded using electrospray ionization tech-
nique with ions given in m/z.
L-Proline, N-Boc-L-a-aspartyl-, 2-methyl 1-(benzyl)
ester (1)
To a solution of L-proline methyl ester hydrochloride
(1.0 g, 6.03 mmol) in DMF was added N-Boc-L-aspartic
acid 4-benzyl ester (1.95 g, 6.03 mmol), N-hydroxyben-
zotriazole (HOBt) (897 mg, 6.64 mmol) and triethylamine
(1.85 mL, 13.28 mmol) dropwise at 0 °C, under N2
atmosphere. After 5 min, EDCI (1.15 g, 6.03 mmol) was
added and continued stirring at room temperature until
TLC indicated consumption of both amine and acid com-
ponents. Reaction mixture was quenched with saturated
NH4Cl and extracted with EtOAc (3 9 50 mL), dried over
Na2SO4, concentrated and purified by flash chromatogra-
phy (7:3 hexane/EtOAc) affording compound 1 (2.141 g,
81 %) as a colorless amorphous solid. Rf 0.45 (1:1 hexane/
1
EtOAc); H NMR (CDCl3) d 1.41 (s, 9H, t-Bu), 2.01 (m,
2H, H-6), 2.19 (m, 2H, H-7), 2.67 (dd, 1H, J = 7, 16, H-2),
2.81 (dd, 1H, J = 5.5, 15.5, H-2), 3.69 (s, 3H, –CO2Me),
3.74 (m, 2H, H-8), 4.50 (m, 1H, H-5), 4.88 (app q, 1H,
H-3), 5.15 (m, 2H, –CH2Ph), 5.40 (s, 1H, –NH), 7.33 (m,
5H, Ph); 13C NMR (CDCl3) d 24.8 (t-Bu), 28.3 (C-6), 29.0
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Med Chem Res (2014) 23:2377–2385
2383
(C-7), 37.5 (C-2), 47.0 (C-8), 52.2 (C-3), 59.0 (C-5), 66.8
(–CH2Ph), 128.2–135.7 (Ph), 170.3 (–CO2Me, –CO2tBu),
172.2 (C-1, C-4); ESI-HRMS [M?Na]? C22H30N2O7Na
calcd for m/z 457.1950, found 457.1928.
Na2SO4, concentrated and purified by flash chromatogra-
phy (3:2 hexane/EtOAc) to afford compound 4 as a col-
orless amorphous solid (2.63 g, 56 %). Rf 0.43 (3:7
1
hexane/EtOAc); H NMR (CDCl3) d 1.42 (s, 9H, t-Bu),
1.81 (m, 2H, H-6), 1.95 (m, 1H, H-7), 2.03 (m, 1H, H-7),
2.70 (dd, 1H, J = 5, 15.5, H-2), 2.93 (dd, 1H, J = 8, 15.5,
H-2), 3.49 (m, 2H, H-9), 3.80 (m, 2H, H-8), 4.13 (m, 1H,
H-5), 4.81 (app q, 1H, H-3), 5.11 (m, 2H, –CH2Ph), 5.37
(bs, 1H, –NH), 7.34 (m, 5H, Ph); 13C NMR (CDCl3) d 24.1
(t-Bu), 27.6 (C-6), 28.1 (C-7), 37.4 (C-2), 47.8 (C-8), 49.5
(C-3), 60.0 (C-5), 64.0 (C-9), 66.8 (–CH2Ph), 80.2 (t-Bu),
128.1–135.3 (Ph), 170.6 (C-1), 170.8 (C-4); ESI-HRMS
[M?H]? C21H31N2O6 calcd for m/z 407.2182, found
407.2159.
Cyclo (L-Pro- L-Asp) 1-(benzyl) ester (2)
Compound 1 (130 mg) was dissolved in formic acid
(2 mL) and allowed to stir at room temperature until TLC
indicated complete consumption of the starting material
(ca. 3–4 h). The volatiles were removed under reduced
pressure and the residue was dissolved in toluene (2 mL).
The resulting solution was heated at reflux for 2 h, con-
centrated and finally purified by flash chromatography (1:1
hexane/EtOAc) to afford compound 2 (104 mg, 80 %) as a
colorless amorphous solid. Rf 0.41 (100 % EtOAc); 1H
NMR (CD3OD) d 1.82 (m, 3H, H-6, H-5), 2.16 (m, 1H,
H-5), 2.84 (t, 2H, J = 5.5, H-8), 3.36 (m, 2H, H-7), 4.14 (t,
1H, J = 6.5, H-3), 4.38 (t, 1H, J = 5.5, H-1), 4.48 (s, 1H,
–NH), 5.03 (m, 2H, –CH2Ph), 7.23 (m, 5H, Ph); 13C NMR
(CD3OD) d 26.0 (C-6), 31.9 (C-5), 38.3 (C-8), 49.1 (C-7),
55.8 (C-1), 57.3 (C-3), 63.0 (–CH2Ph), 131.9–139.6 (Ph),
169.5 (C-2, C-4), 174.2 (C-9); ESI-HRMS [M?Na]?
C16H18N2O4Na calcd for m/z 325.1164, found 325.1152.
(3S, 8aS)-Pyrrolo[1,2-a]pyrazine-3-acetic acid, 2-[1,1-
dimethylethoxy)carbonyl] 4-oxooctahydro, benzyl ester (5)
To a solution of compound 4 (1.07 g, 2.66 mmol) in DCM
(8 mL) was added Et3N (1.09 mL, 7.94 mmol) dropwise at
0 °C. After 15 min, MsCl (0.288 mL, 1.4 mmol) was
added dropwise at 0 °C under N2 atmosphere over a period
of 10 min. After completion of starting material, as indi-
cated by TLC, the reaction mixture was quenched with ice
water and extracted with EtOAc (3 9 30 mL), dried over
Na2SO4, concentrated, and directly used for the next step
without purification. To the crude mesylate (1.281 g,
2.646 mmol) in DMF was added Cs2CO3 (0.25 g,
7.94 mmol) and subjected to reflux for 2 h. The reaction
mixture was quenched with water and extracted with
EtOAc (3 9 50 mL), dried over Na2SO4, concentrated,
and purified by flash chromatography (3:2 hexane/EtOAc)
to afford compound 5 (0.64 g, 63 %) as a colorless amor-
phous solid. Rf 0.39 (3:7 hexane/EtOAc); 1H NMR
(CDCl3) d 1.46 (s, 9H, t-Bu), 1.73 (m, 2H, H-6), 1.99 (m,
2H, H-5), 2.91 (m, 2H, H-8), 3.14 (d, 1H, J = 15.5, H-2),
3.48 (m, 3H, H-7, H-2), 4.39 (m, 1H, H-3), 4.64 (m, 1H,
H-1), 5.09 (m, 2H, -CH2Ph), 7.34 (m, 5H, Ph); 13C NMR
(CDCl3) d 22.4 (C-6), 28.3 (t-Bu), 30.0 (C-5), 37.1 (C-8),
43.8 (C-7), 45.4 (C-2), 53.2 (C-1), 57.9 (C-3), 66.5
(–CH2Ph), 80.8 (t-Bu), 128.1–135.5 (Ph), 153.8 (–CO2tBu),
166.0 (C-4), 170.7 (C-9); ESI-HRMS [M?H]? C21H29N2O5
calcd for m/z 389.2076, found 389.2055.
Cyclo (L-Pro- L-Asp) (3)
Compound 2 (104 mg) was dissolved in ethanol (2 mL)
and then added Pd/C 10 % (50 mg). The reaction mixture
was stirred under hydrogen atmosphere until consumption
of compound 2, as indicated by TLC. The reaction mixture
was passed through the Celite pad and the filtrate was
concentrated and precipitated with ether (5 mL) to afford
compound 3 (29 mg, 39 %) as a colorless amorphous solid.
1H NMR (CD3OD) d 1.87 (m, 3H, H-6, H-5), 2.21 (m, 1H,
H-5), 2.52 (dd, 1H, J = 7.5, 17, H-8), 2.82 (dd, 1H, J = 4,
17, H-8), 3.44 (m, 2H, H-7), 4.16 (app t, 1H, H-3), 4.32 (m,
1H, H-1), 7.23 (s, 1H); 13C NMR (CD3OD) d 23.4 (C-6),
29.3 (C-5), 37.1 (C-8), 46.5 (C-7), 53.8 (C-1), 60.3 (C-3),
167.7 (C-2, C-4), 171.9 (C-9); ESI-HRMS [M?Na]?
C9H12N2O4Na calcd for m/z 235.0694, found 235.0685.
L-Prolinol, N-Boc-L-a-aspartyl-, 1-(benzyl) ester (4)
To a solution of N-Boc-L-aspartic acid 4-benzyl ester
(3.76 g, 11.63 mmol) in DMF (10 mL) was added BOP
(6.17 g, 13.96 mmol), HOBt (1.88 g, 13.96 mmol), and
DIPEA (3.97 mL, 23.27 mmol). After 5 min, (S)-(?)-
prolinol (1.53 g, 15.12 mmol) was added and the reaction
mixture was stirred at room temperature until the con-
sumption of starting materials, as indicated by TLC. The
reaction mixture was quenched with saturated NaHCO3
and extracted with EtOAc (3 9 50 mL), dried over
(3S, 8aS)-Pyrrolo[1,2-a]pyrazine 4-oxooctahydro-3-
acetic acid (6)
To the compound 5 (106 mg, 0.27 mmol) in EtOH (4 mL)
was added Pd/C 10 % (55 mg) and the reaction was stirred
under H2 atmosphere. After completion of reaction, as
indicated by TLC, the reaction mixture was passed through
the Celite pad and concentrated. To the filtrate in DCM
(4 mL), trifluoroacetic acid was added (526 lL, 6.87 mmol)
123

2384
Med Chem Res (2014) 23:2377–2385
CLSI, Clinical and Laboratory Standards Institue (2010) Performance
standards for antifungal disk diffusion susceptibility testing of
non-dermatophyte filamentous fungi; informational supplement-
first edition. CLSI document M51-A. Clinical and Laboratory
Standards Institute, Villanova, PA
CLSI, Clinical and Laboratory Standards Institute (2008) Reference
method for broth dilution antifungal susceptibility testing of
filamentous fungi, as the document is M38-A2, Wayne, PA
CLSI, Clinical and Laboratory Standards Institute (2009) Method for
antifungal disk diffusion susceptibility testing of yeasts;
approved guideline, 2nd ed., M44-A2 Clinical and Laboratory
Standards Institute, Wayne, PA
and stirred at room temperature. After completion of the
reaction as indicated by TLC, the reaction mixture was
concentrated and the residue was washed with DCM
(5 9 5 mL) to afford compound 6 (54 mg, quant.) as a
1
colorless amorphous solid. H NMR (CD3OD) d 1.57 (m,
1H, H-6), 1.81 (m, 1H, H-6), 1.97 (m, 1H, H-5), 2.16 (m, 1H,
H-5), 2.95 (m, 2H, H-8), 3.08–3.56 (m, 4H, H-2, H-7), 3.77
(m, 1H, H-3), 4.17 (m, 1H, H-1); 13C NMR (CD3OD) d 23.3
(C-6), 30.7 (C-5), 31.4 (C-8), 45.2 (C-7), 47.0 (C-2), 52.9
(C-1), 55.8 (C-3), 165.0 (C-4), 173.9 (C-9); ESI-HRMS
[M?H]? C9H15N2O3 calcd for m/z 199.1082, found
199.1076.
CLSI, Clinical and Laboratory Standards Institute (2012a) Methods
for dilution antimicrobial susceptibility tests for bacteria that
grow aerobically; approved standard-ninth edition. CLSI docu-
ments M07-A9. Wayne, PA
CLSI, Clinical and Laboratory Standards Institute (2012b) Performance
standards for antimicrobial disk susceptibility tests; approved
standard-eleventh edition. CLSI documents M02-A11. Wayne, PA
CLSI, Clinical and Laboratory Standards Institute (2012c) Reference
method for broth dilution antifungal susceptibility testing of
yeasts, as the document is M27-S4, Wayne, PA
Conclusion
In summary, this is the first report demonstrating antimi-
crobial evaluation of cyclo(L-Pro- L-Asp) DKP against
broad-spectrum bacteria and fungi. A diminished antimi-
crobial activity of the 2-ketopiperazine analog of cyclo
(L-Pro- L-Asp) against all test organisms indicates the sig-
nificance of peptide bond in cyclo(L-Pro- L-Asp). The most
potent antifungal activity exhibited by cyclo(L-Pro- L-Asp)
was against T. rubrum (MIC 2 lg/mL). Based on the
comparison with known antifungal drugs, the mode of
interaction of DKPs with fungal cells, of whether they are
surface interacting molecules or target molecules inside the
cells, warrants further investigation.
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& Technology (DST), India, Grant SR/FT/CS-45/2010 is gratefully
acknowledged.
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