Multicomponent Approach to a Library of N-Substituted γ-Lactams

Article abstract of DOI:10.1021/acscombsci.8b00147

The γ-lactam motif is often found in naturally occurring compounds with diverse biological activities. We prepared a 28-member library of N-substituted γ-lactams following a single-pot, three-component Ugi reaction comprising bifunctional building block,

Full text of DOI:10.1021/acscombsci.8b00147

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Article
Multicomponent approach to a library of N-substituted #-lactams
Josipa Su# Sajko, Vanja Ljolji# Bili#, Ivan Kosalec, and Ivanka Jeric
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00147 • Publication Date (Web): 18 Dec 2018
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Multicomponent approach to a library of N-
substituted -lactams
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Josipa Suć Sajko,a Vanja Ljoljić Bilić,b Ivan Kosalec,b and Ivanka Jerića*
a Ruđer Bošković Institute, Division of Organic Chemistry and Biochemistry, Bijenička cesta 54,
10000 Zagreb, Croatia.
b Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb,
Schrottova 39, 10000 Zagreb, Croatia
KEYWORDS: -lactams, multicomponent reactions, Ugi reaction, antibiofilm activity.
ABSTRACT: The γ-lactam motif is often found in naturally occurring compounds with diverse
biological activities. We prepared a 28-member library of N-substituted -lactams following a
single-pot, three-component Ugi reaction comprising bifunctional building block, L-glutamic acid
methyl ester. The reaction tolerates structurally diverse carbonyl and isocyanide components
providing a robust access to functionalized γ-lactams. Antimicrobial susceptibility testing,
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including agar well diffusion assay, serial microdilution broth assay and antibiofilm activity
testing, identified a potent compound with antibiofilm activity against S. aureus ATCC 6538.
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O
O
R1
O
R2
N
H
N
H2N
OMe
60 °C
TFE
R3
R1
R2
O
MeO
R3 NC
O
O
OH
-substituted -lactams
N

28 examples
yields up to 84 %
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INTRODUCTION
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The pyrrolidin-2-one, also known as -lactam ring, is the core structure of a large number of
natural and non-natural compounds covering a broad spectrum of biological activities.1 The
synthesis of substituted -lactams is an important goal, because of their use as key intermediates
in the synthesis of biologically and pharmaceutically relevant molecules.2 Important examples of
these -lactams include the proteasome inhibitor lactacystin (I),3 (-)-clausenamide (II) for the
treatment of Alzheimer’s disease,4 (-)-dysibetaine (III) and anantine (IV) exhibit cytotoxic,
antitumor and anti-inflammatory activities5,6 while (±)-rolipram (V) is a selective inhibitor of
phosphodiesterase type IV, anti-inflammatory agent and antidepressant (Figure 1).7 Compounds
containing -lactams have direct applications in the treatment of epilepsy,8 HIV,9
neurodegenerative diseases and depression.10,11 Many approaches have been developed for the
preparation of -lactam structures. The most widely used method is cyclization by amide formation
between a carboxylic group and an amine or its precursor. Cyclization can also be performed by
intramolecular N-alkylation of an amide or by C–C bond formation.12 Beside these cyclization
strategies, several (3+2) and (4+1) cycloaddition/annulation reactions leading to the -lactam
structure have also been designed.13
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Furthermore, recognizing the potential for discovery of novel bioactive small molecules based
upon the -lactam core structure, several research groups have presented interesting
multicomponent or cascade approaches towards functionalized -lactams.14-16 Multicomponent
reactions (MCRs) were found to be an excellent tool to rapidly access a large and versatile drug-
like chemical space to address the corresponding biological space.17
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O
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NH
HO
O
OH
O
O
OH
NHAc
S
O
H
8
N
OH
HN
N
9
OH
O
OH
O
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I
II
III
O
NH
N
N
O
N
H
O
OMe
IV
V
Figure 1. Biologically active -lactam structures.
Among these reactions, isocyanide based multicomponent reactions (IMCRs), like Passerini and
Ugi reaction, are particularly popular due to the carbenic reactivity of isocyanides, wide substrate
scope and mild reaction conditions.18 The classical Ugi reaction combines four components; an
amine, an aldehyde or ketone, a carboxylic acid and an isocyanide, in a one-pot manner to produce
an α-acylaminoamide. This process, known as the Ugi four-component reaction (U-4CR), has
found widespread application in both, diversity and target oriented organic syntheses.19 In 1996
Ugi reported an effective variation of the U-4CR as a 5-centre 4-component reaction (U-5C-4CR)
with very high yields and excellent stereoselectivity.20 α-Amino acids serve as bifunctional starting
materials in the U-5C-4CR instead of the amine and acid components in the familiar U-4CR. They
react with equimolar amounts of aldehyde and isocyanide as well as with the alcohol that also
serves as a solvent.20,21 The main advantage of bifunctional substrates in IMCRs is a smooth access
to novel, functionalized cyclic scaffolds.22 A convenient and versatile synthesis of N-substituted
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-lactams using convergent Ugi reaction was exploited by several groups.23-25 Gunawan et al.
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described solid-phase synthesis of N-substituted -lactams utilizing resin-bound glutamic acid, but
with very limited substrate scope.25 In this report we used C-terminal protected L-glutamic acid as
a bifunctional building block in the 4-centre 3-component Ugi reaction (U-4C-3CR) with various
carbonyls and isocyano-components. The advantage of this approach is that only three components
are involved in one reaction step, thus enabling a smooth access to a library of diverse N-
substituted -lactams.
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RESULTS AND DISCUSSION
Chemical Synthesis
Our initial reaction was conducted with L-glutamic acid methyl ester (1), p-nitro benzaldehyde
2{1} and cyclohexyl isocyanide 3{1}. First reaction performed in dichloromethane didn't give a
corresponding product, probably due to poor solubility of the reactants (Table 1, Entry 1). The
same reaction performed in dichloromethane/methanol (10:1, v/v) mixture gave, after 48 h, desired
N-substituted -lactam product 4{1,1}, although in low yield (28 %, Entry 2). Encouraged by this
result, we tested different polar solvents, commonly used for the Ugi reaction (Table 1, Entries 3-
8). Running the reaction in methanol afforded the product in a disappointing yield (22 %, Entry
3), while heating up the reaction mixture at 60 °C for 24 h improved the yield to 47 % (Entry 4).
Next, we tested trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) as solvents at the room
temperature and at elevated temperature for 24 and 72 hours. Both, TFE and HFIP at 60 °C proved
to be suitable for this type of the Ugi reaction (75 % and 84 %, respectively, after 24 h; Entries 6
and 8). For economic reasons, the less expensive TFE was used in all further reactions.
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Table 1. Screening of reaction conditions for the Ugi 3-component reaction.
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O
NO2
O
H
H2N
OMe
temp.
O2N
O
H
2{1}
3{1}
N
solvent
N
O
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O
O
OH
MeO
CN
1
4
{1,1}
Entry
Solvent
DCM
T/ °C Time/h
Yield/%b
-
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2
3
4
5
6
7
8
RT
24
DCM/MeOH (10:1) RT
48
28
MeOH
MeOH
TFE
RT
60
24
22
48
47
RT
60
24/72
24/72
24
68/70
75/83
80
TFE
HFIP
HFIP
RT
60
24
84
[a]
Reactions were performed with 0.17 mmol of p-nitro benzaldehyde, 0.19 mmol of H-Glu-OMe and 0.19 mmol of cyclohexyl
isocyanide. [b] Isolated yields.
With the optimized conditions in hand, we set out to test the scope of the protocol by utilizing
different carbonyl components (Chemset 1, Figure 2) and isocyanides (Chemset 2, Figure 2). First
batch of Ugi-3CR was conducted with L-glutamic acid methyl ester, p-nitro benzaldehyde and
different commercially available and synthesized isocyanides (see Supporting Information), and
results are summarized at Figure 3.
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NO2
Cl
OMe
4
5
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7
8
O
O
O
O
O
O
O
O
O
9
2
2
2
2
2
2
2
2
2
{9}
{1}
{2}
{3}
{4}
{5}
{6}
{7}
{8}
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O
O
O
O
O
O
BocN
BocN
BocHN
BocHN
BocHN
O
O
O
O
O
O
O
O
O
O
O
O
2
2
2
2
2
2
2
{16}
{10}
{11}
{12}
{13}
{14}
{15}
2
Chemset 1 Diversity reagents {1-16}
O
O
OMe
CN
CN
OMe
CN
CN
CN
CN
3
3
3
3
3
3
{6}
{1}
{2}
{3}
O
{4}
{5}
O
O
OMe
OMe
OMe
CN
MeO
OMe
CN
CN
CN
3
3
3
3
{10}
{7}
{8}
{9}
3
Chemset 2 Diversity reagents {1-10}
Figure 2. Chemsets selected for the library of N-substituted -lactam products: carbonyl
components (Chemset 1), isocyanides (Chemset 2).
Reactions performed with commercially available isocyanides 3{2-4} afforded corresponding
products in good to very good yields (53-83 %, products 4{1,2-4}). Next, we tested aromatic
isocyanides 3{5-7}, and obtained -lactams in fair yield (57 % and 54 % for compounds 4{1,6},
and 4{1,7}, respectively), with the exception of compound 4{1,5}, obtained in only 24 % yield.
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Possible explanation for this result can be observed low stability of the corresponding isocyanide.
Finally, we examined amino acid-derived racemic isocyanides 3{8-10} and obtained -lactam
scaffolds embedded into the tripeptide structures, in satisfactory yields (57-80 % for compounds
4{1,8-10}, Figure 3). Since the best yield is achieved in the reaction with 1,1,3,3-tetramethylbutyl
isocyanide 3{3} (Figure 3, product 4{1,3}), it was used in the second batch of Ugi-3CR during the
screening of different carbonyl components.
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Comparison of the Ugi reactions performed with benzaldehyde 2{2} (product 4{2,3}), p-Cl
benzaldehyde 2{3} (product 4{3,3}), and p-OMe benzaldehyde 2{4} (product 4{4,3}), revealed
that unsubstituted aromatic aldehyde and an aldehyde bearing electron withdrawing substituent
furnished corresponding Ugi products in very good yields (82 %), compared to aromatic aldehyde
bearing electron donating substituents (40 %, Figure 3). Additionally, aliphatic aldehydes, 2-
methyl-pentanal 2{5}, and cyclohexylcarboxaldheyde 2{6} furnished corresponding products in
satisfactory yields (62-89 %, products 4{5-6,3} and 4{6,8}). Finally, three ketones were used in
the Ugi reaction; product 4{7,3} was obtained with acetone 2{7} in rather low yield (24 %), while
acetophenone 2{8} failed to give the expected product 4{8,3} presumably due to steric reasons.
Contrary to that, reaction with cyclohexanone 2{8} gave the desired product 4{9,3} in very good
yield (74 %). Furthermore, we used chiral N-terminally protected amino aldehydes derived from
valine 2{10}, leucine 2{11}, phenylalanine 2{12}, and proline 2{13} to introduce additional
functionality into the Ugi product. As presented at the Figure 2, all performed reactions furnished
corresponding Ugi products in moderate to very good yields (22-84 %, products 4{10-13,3} and
4{13,1}). Comparison of obtained results revealed that steric hindrance of the amino aldehyde
have impact on the yield of the Ugi reaction. Nice examples are Ugi products 4{10,3} (39 %) and
4{13,3} (22 %) with branched  carbon atoms in comparison with sterically less demanding
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4{11,3} (75 %) and 4{12,3} (84 %). However, the influence of isocyanide cannot be neglected, as
apparent by comparing product 4{13,3} (22 %) and 4{13,1} (58 %), differing in the nature of
isocyano component. Finally, we introduced Garner's aldehyde 2{14}[26] and obtained Ugi product
4{14,3} in fair yield (41 %), while a 63 % yield was achieved in the reaction with cyclohexyl
isocyanide, 4{14,1}. To further widen substrate scope of carbonyl compounds, we performed two
reactions with sugar derived aldehydes. Bis-isopropylidene protected D-galactose-derived
aldehyde 2{15} gave Ugi product 4{15,3} in very good yield (72 %), while bis-isopropylidene
protected D-fructose-derived aldehyde 2{16} furnished Ugi product 4{16,3} in somewhat lower,
but satisfactory yield (59 %).
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O
R1
H2N
O
O
R2
N
H
OMe
N
O
60 °C
TFE
R3
O
CN R3
R1
R2
MeO
2
3
O
OH
8
1
4
9
NO2
NO2
NO2
NO2
NO2
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O
O
O
O
O
O
OMe
H
H
N
H
H
N
H
N
N
N
N
N
MeO
N
N
O
N
O
O
O
O
O
O
O
O
O
O
OMe
OMe
OMe
OMe
OMe
4
4
4
4
4
{1,6} (57 %)
{1,2} (53 %)
{1,3} (83 %)
{1,4} (75 %)
{1,5} (24 %)
NO2
NO2
NO2
NO2
O
O
NO2
H
O
O
O
O
O
H
H
N
H
N
N
N
N
N
MeO
MeO
N
MeO
N
O
O
O
O
O
O
MeO
O
O
OMe
OMe
OMe
OMe
4
{1,7} (54 %)
4
Cl
4
4
{1,10} (63 %)
{1,8} (57 %)
{1,9} (80 %)
OMe
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
N
N
N
O
N
N
O
MeO
O
MeO
O
O
O
MeO
MeO
MeO
O
O
O
O
4
4
4
4{5,3} (62 %)
4{6,3} (89 %)
{2,3} (82 %)
{3,3} (40 %)
{4,3} (82 %)
O
O
O
OMe
H
H
N
H
N
H
N
O
N
N
O
N
O
N
O
O
N
O
O
O
O
MeO
MeO
MeO
MeO
O
4
4
4
4
{9,3} (74 %)
{6,8} (73 %)
{7,3} (24 %)
{8,3} (n.o.)
BocN
H
N
BocN
BocHN
BocHN
BocHN
O
O
O
O
H
N
O
H
N
H
N
H
N
N
N
N
O
N
N
O
MeO
O
O
O
O
O
MeO
MeO
MeO
MeO
O
O
O
4
4
4
4
4
{13,1} (58 %)
{10,3} (39 %)
{11,3} (75 %)
{12,3} (84 %)
{13,3} (22 %)
O
O
O
O
O
O
O
O
BocN
BocN
O
N
O
H
N
O
O
O
O
O
O
H
N
H
N
H
N
N
N
N
O
O
O
O
MeO
MeO
MeO
MeO
O
O
O
O
4
4
4
4
{16,3} (59 %)
{14,3} (41 %)
{14,1} (63 %)
{15,3} (72 %)
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Figure 3. Scope of the Ugi reaction with different isocyanides and oxo-compounds.
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The formation of new stereocentre is inherent to the Ugi reaction, and is generally accepted that
the nature of amine component dictates the stereoselectivity of the reaction.[27] Since amine and
acid component are the same in all Ugi reactions performed within this work, any differences in
stereoselectivity of the reaction can be attributed to the carbonyl and isocyanide components. All
Ugi products are isolated as inseparable mixture of diastereoisomers, and the inspection of the
NMR spectra revealed that there is virtually no diastereoselectivity or is very modest. The best
result, 77:23 d.r. is obtained with Ugi product 4{1,1}. Although it is expected that chiral aldehydes
can induce some stereoselectivity, their influence is generally not significant under the conditions
performed in this work.
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The currently accepted mechanism of the U-4C-3CR starts with condensation of the amino acid
with the carbonyl component to form the corresponding protonated Schiff’s base I. Addition of
the isocyanide then produces the cyclic O-acyl-imide II. Final step is the Mumm rearrangement to
form the five-membered ring III (Scheme 1).[28]
O
R2
+
-
C N R2
+
R1
H2N
H
N
C
MeOH
O
R1
H
O
R1
H
O
OMe
N
H
O
HN
OMe
OMe
-O
O
O
O
I
HO
R2
N
R1
O
H
O
R1
O
N
R2
N
HN
O
MeO
O
O
MeO
II
III
Scheme 1. Proposed mechanism of the U-4C-3CR
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Biological Screening
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With a library of N-substituted -lactams in-hand and having in mind that -lactams cover a
broad spectrum of biological activities, we decided to test antimicrobial activity of selected
compounds and thus performed an agar well diffusion assay, a serial microdilution broth assay and
antibiofilm activity testing. Along with compounds prepared by the 3-component Ugi reaction, we
also included compounds 5-8 (Figure 4) obtained by acid hydrolysis of compounds 4{11,3},
4{12,3}, 4{13,1}, and 4{14,1}, respectively. Altogether, 28 compounds were investigated, except
for compounds 4{1,5}, 4{1,7}, 4{7,3}, and 4{8,3}, which were unstable or not obtained. Results
of all performed assays were calculated as the mean of three independent experiments. No
antimicrobial activity was observed in an agar well diffusion assay and a serial microdilution broth
assay in tested concentrations (Table S1, Supporting Information). Within the initial antibiofilm
screening, activity was observed for compound 4{13,1} (inhibition less than 50 %, data not
shown), so its activity during biofilm formation was investigated in more details (Table 2). The
MBFIC50 and MBFIC90 values represent the lowest compound dilution at which bacterial growth
during biofilm formation was inhibited for 50 % and 90 %, in comparison to untreated control. It
turned out that compound 4{13,1} shows potent antibiofilm formation activity against S. aureus
ATCC 6538 with MBFIC50=1.906 µg/ml and MBFIC90=484.172 µg/ml (Figure 5).
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HCl x
H
H2N
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HCl H2N
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MeO
MeO
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O
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O
HCl x HN
H
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HCl x
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MeO
MeO
O
O
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Figure 4. Compounds obtained by acid hydrolysis of compounds 4{11,3}, 4{12,3}, 4{13,1}, and
4{14,1}.
Biofilms are defined as aggregated microorganism communities attached to surfaces and
embedded in a self-produced matrix.29 In this, actually defensive state, bacteria exhibit slowed
metabolism and display up to 1000-fold enhanced resistance to antibiotic treatment. According to
National Institutes of Health, bacterial biofilms are responsible for over 80 % of infections found
in the human body.30 Naturally occurring biofilm inhibitors act as sources of inspiration in the
design of small molecules with potential antibiofilm formation activity. Libraries of 2-
aminoimidazoles, benzimidazoles or indole–triazole-amide analogs, along with brominated
furanone analogs, N-acyl homoserine lactone analogs and peptide-based compounds have been
prepared and tested.29,31,32 Based of these findings, our understanding of underlying molecular
mechanisms of bacterial biofilm formation has increased, but to date, no antibiofilm drug has been
registered and is in clinical use. This significantly hampered the treatment of biofilm-related
infections especially in health-related environments. According to a recent study, three different
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-alkylidene--lactams showed biofilm inhibition on Streptococcus mutans, Enterococcus faecalis,
and Candida glabrata.33 It therefore seems meaningful to further explore the potential of N-
substituted -lactams to modulate bacterial virulence, through biofilm eradication. So, our next
step is to design and develop a focused library of compounds based on structure of 4{13,1}, where
proline-derived aldehyde will be replaced by its analogous along with different isocyanides.
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Table 2. Anti-biofilm activity of compound 4{13,1} on S. aureus ATCC 6538.
a
a
b
b
Compound MBIFC50
MBFIC90
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MMBIC50
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MMBIC90
/µg/ml
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/µg/ml
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>1250
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sulphate
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6280.584
[a] MBFIC= minimal biofilm-forming inhibition concentration [b] MMBIC= minimal mature-biofilm inhibition
Figure 5. Compound 4{13,1} shows potent anti-biofilm formation activity against S. aureus
ATCC 6538 (MBFIC50=1.906 µg/ml and MBFIC90=484.172 µg/ml).
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We have prepared a diverse selection of N-substituted -lactams in an efficient single-step
procedure, utilizing a bifunctional α-carboxy protected L-glutamic acid with diverse isocyanides
and aldehydes or ketones. Functionalized -lactones are obtained in up to 84 % yield and their
antimicrobial activity was tested. Even though no antibacterial activity was shown within the agar
well diffusion assay and serial two-fold microdilution broth assay, the observed antibiofilm
formation potency of compound 4{13,1} for S. aureus ATCC 6538 gives impetus for further
antibiofilm investigations, especially regarding gram-positive bacteria.
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Experimental procedures
General Procedure for the Ugi-5-center-4-component reaction
To a glass vial containing 1 M solution of H-Glu-OMe (1) (0.19 mmol; 1,1 eq) in TFE were
added oxo-compound (0.17 mmol) and the isocyanide (0.19 mmol; 1,1 eq). With all reactants
added, the solution was allowed to stir at 60° C for 24 h. The reactions were concentrated under
reduced pressure and reaction mixtures were purified by flash column chromatography.
Representative example of the Ugi product:
(2R)-methyl 1-(1-(4-nitrophenyl)-2-oxo-2-(2,4,4-trimethylpentan-2-ylamino)ethyl)-5-oxo
pyrrolidine-2-carboxylate 4{1,3}.
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Colorless oil (68 mg; 83 %); dr 58:42. H NMR (600 MHz, CDCl3) δ / ppm 8.20 - 8.18 (Har,
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Ph, 3a, 3b, m, 2H), 7.62 – 7.60 (Har, Ph, 3a, m, 1H), 7.58 – 7.56 (Har, Ph, 3b, m, 1H), 5.68 (H1,
3a, s, 1H), 5.57 (H1, 3b, s, 1H), 4.68 – 4.65 (H2, 3a, m, 1H), 4.28 (H2, 3b, m, 1H), 3.74 (OMe, 3a,
s, 3H), 3.36 (OMe, 3b, s, 3H), 2.59 (H4, 3a, m, 1H), 2.50 – 2.19 (H3, 3a, 3b, m, 2H), 2.12 – 1.98
(H4, 3b, m, 1H), 1.44 (H8, H9, 3a, m, 4H), 1.41 (H8, H9, 3b, m, 4H), 1.39 (H6, 3a, 3b, m, 2H),
0.96 (CH3, t-Bu, 3a, s, 9H), 0.92 (CH3, t-Bu, 3b, s, 9H).
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13C NMR (151 MHz, CDCl3) δ / ppm 176.1 (CO, 3a), 175.6 (CO, 3b), 172.8 (CO, 3a), 172.6
(CO, 3b), 166.6 (CO, 3a), 165.9 (CO, 3b), 147.9 (Car, 3a), 147.8 (Car, 3b), 142.8 (Car, 3a), 141.1
(Car, 3b), 130.8, 129.2, 123.8, 123.6 (CHar, 3a, 3b), 59.9 (C1, 3a), 59.4 (C1, 3b), 58.8 (C2, 3a),
58.7 (C2, 3b), 56.0 (C6, 3a), 55.9 (C6, 3b), 52.5 (OMe, 3a), 52.2 (OMe, 3b), 52.2 (C5, 3a), 51.9
(C5, 3b), 31.6 (C7, 3a), 31.6 (C7, 3b), 31.6, 31.5, 31.4, 31.4, 30.9, 30.9 (CH3, t-Bu, 3a, 3b), 29.2
(C4, 3a), 29.1 (C4, 3b), 28.8, 28.6, 28.5, 28.4 (C8, C9, 3a, 3b), 24.6 (C3, 3a), 24.2 (C3, 3b).
MS-ESI: m/z 432.0 [M-H]-; m/z 434.3 [M+H]+; m/z 456.3 [M+Na]+; m/z 889.5 [2M+Na]+.
HRMS: calcd for C22H31N3O6 [M+H]+ 434.2291, found 434.2307.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at:
General methods, synthetic procedures, characterization data, and copies of 1H, 13C and HRMS
spectra, antimicrobial susceptibility testing and anti-biofilm activity screening. (PDF)
AUTHOR INFORMATION
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Corresponding Author
*E-mail: ijeric@irb.hr.
Author Contributions
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The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
Acknowledgements
We are grateful for financial support from the Croatian Science Foundation, Grant number 3102.
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