A Facile Synthesis of Hydroxamic Acids of Nα-Protected Amino Acids Employing BDMS, a Study of Their Molecular Docking and Their Antibacterial Activities

Full text of DOI:10.1080/00304948.2019.1579039

Organic Preparations and Procedures International
The New Journal for Organic Synthesis
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A Facile Synthesis of Hydroxamic Acids of Nα-
Protected Amino Acids Employing BDMS, a Study
of Their Molecular Docking and Their Antibacterial
Activities
K. Uma, H. S. Lalithamba, V. Chandramohan & K. Lingaraju
To cite this article: K. Uma, H. S. Lalithamba, V. Chandramohan & K. Lingaraju (2019): A Facile
Synthesis of Hydroxamic Acids of Nα-Protected Amino Acids Employing BDMS, a Study of Their
Molecular Docking and Their Antibacterial Activities, Organic Preparations and Procedures
International, DOI: 10.1080/00304948.2019.1579039
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Organic Preparations and Procedures International, 0:1–14,
# 2019 Taylor & Francis Group, LLC
ISSN: 0030-4948 print / 1945-5453 online
DOI: 10.1080/00304948.2019.1579039
A Facile Synthesis of Hydroxamic Acids of
Na-Protected Amino Acids Employing BDMS,
a Study of Their Molecular Docking and
Their Antibacterial Activities
K. Uma,1 H. S. Lalithamba,1 V. Chandramohan,2 and
K. Lingaraju3
1Department of Chemistry, Siddaganga Institute of Technology, B.H. Road,
Tumakuru-572 103, Karnataka, India
2Department of Biotechnology, Siddaganga Institute of Technology, B.H.
Road, Tumakuru-572 103, Karnataka, India
3Department of Biotechnology, Tumkur University, B.H. Road, Tumakuru-
572 103, Karnataka, India
Hydroxamic acids have received much attention as biologically active compounds.
Synthetic hydroxamic acids enhance the growth of plant sources and improve the soil
quality, act as antibiotics, cell division agents or tumor inhibitors.1 Hydroxamic acids
were found to be histone deacetylase (HDAC) inhibitors2 and the functionality of
hydroxamic acid is an important structural component in bioactive agents such as anti-
bacterial, antifungal and anticancer agents.3 The one-pot oxidative transformation of
aldehydes into hydroxamic acids using an aqueous solution of hydroxylamine4 has been
reported. Acylation of carboxylic acids with hydroxylamine using coupling reagents
like cyanuric chloride,5 propylphosphonic anhydride6 and N,N0-carbonyldiimidazole7
can be found in the literature. Economical ways to obtain hydroxamic acid derivatives
by the reaction of hydroxylamine salt with acid chlorides and esters8 have been
described. The reported methods for the preparation of hydroxamic acids include the
reaction of activated carboxylic acids with O/N- protected hydroxylamines9 and MgO
mediated preparation of Fmoc-protected amino acid hydroxamates from acid chlor-
ides.10 Suresh Babu et al., achieved the synthesis of Na-protected amino/peptide
hydroxamic acids mediated by (1-cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethyla-
mino-morpholino-carbenium hexafluorophosphate (COMU)11 and 1-propanephosphonic
acid cyclic anhydride (T3P) as proficient promoters for the Lossen rearrangement
(LR).12 These hydroxamic acids are vital precursors for the LR to obtain ureas, carba-
mates and thiocarbamates.
Received June 20, 2018; in final form November 17, 2018.
Address correspondence to H. S. Lalithamba, Department of Chemistry, Siddaganga Institute of
Technology, B.H. Road, Tumakuru-572 103, Karnataka, India. E-mail: lalithambasit@yahoo.co.in
This article is dedicated to Dr. Sree Sree Sree Shivakumara Mahaswamiji, Siddaganga Math,
Tumakuru, Karnataka, India.
Color versions of one or more of the figures in the article can be found online at www.tandfon-
line.com/uopp.
1

2
Lalithamba et al.
In this study, we report the application of bromodimethylsulfonium bromide
(BDMS) for the synthesis of hydroxamic acids. The reagent BDMS is readily available
and has numerous applications in synthetic chemistry. According to a literature survey,
this reagent acts as a catalyst for numerous organic transformations and even as a bro-
minating reagent.13 The one-pot synthesis of a-aminophosphonates from aldehyde,
amine and phosphite was established from the catalytic activity of BDMS.14 Nitration
of phenols with tetrabutylammonium nitrite in the presence of BDMS was reported.15
BDMS was also employed for the synthesis of a-bromo-b-keto esters and a-bromoe-
nones, from their corresponding keto esters and enones respectively.16 One-pot multi-
component synthesis of functionalized piperidines using BDMS was reported.17 BDMS
was employed as an efficient and regioselective reagent for aromatic and heteroaromatic
thiocyanation of ketones and b-dicarbonyl compounds in the presence of ammonium
thiocyanate.18 BDMS accelerated the synthesis of a xanthine,19 acted as a brominating
reagent for the conversion of anthracene into 9,10-dibromoanthracene20 and promoted
the ring opening reactions of epoxides and aziridines at room temperature.21 Khan and
others have developed the synthesis of 2,3-unsaturated O-glycosides using BDMS as
pre-catalyst.22 BDMS catalyzed the three-component synthesis of a-amino amidines
from aldehydes, amines and isocyanides.23 BDMS was used as a reagent for the synthe-
sis of unsymmetrical ureas.24 Further, it was found to be a useful reagent for the con-
version of primary amides and aldoximes into nitriles.25
However, there are no reports for the synthesis of Na-protected hydroxamic acids
employing BDMS in peptide chemistry. In the present study BDMS was used for the
synthesis of ureas and carbamates in one pot through LR by the transformation of the
hydroxamic acid into its isocyanate26 and then coupling with an amine or alcohol.
Isocyanates can be obtained via the Hofmann rearrangement but the rearrangement is
not well-suited with amino acid chemistry due to epimerization problems.27 The LR of
hydroxamic acids to isocyantes is also well known. Reagents such as N,N1-dicyclohexyl
carbodiimide,28 N,N1-carbonyldiimidazole29 and cyanuric chloride30 have been
employed for the formation of isocyanates via LR. Thalluri and others reported the
ethyl 2-cyano-2-(4-nitrophenylsulfonyloxyimino) acetate-mediated LR for the synthesis
of hydroxamic acids and ureas from carboxylic acids.31 Some of the protocols men-
tioned here suffer due to reagent instability, longer reaction times, low yields or mul-
tiple steps; some could not be extended to N-Boc/Cbz-protected amino acids. The
disadvantage of these reported protocols is mainly the accumulation of isocyanate
before the complete activation of hydroxamic acid leading to the formation of by-prod-
uct. Such a problem can be solved by the use of an efficient promoter for LR.
Herein, we report an efficient protocol for the synthesis of hydroxamic acids from
Na-Fmoc/Cbz/Boc amino acids using BDMS as an activating agent. BDMS was utilized
for the formation of intermediate isocyanates and conversion of the latter into ureas and
carbamates in one pot. The prepared hydroxamic acids were subjected to in silico
molecular docking studies32 and in vitro antibacterial activities.
For the synthesis of Na-protected hydroxamic acids (2, Scheme 1), a protected
amino acid was dissolved in acetonitrile. N-methylmorpholine (NMM) and BDMS were
added at 0ꢀC and the solution was stirred for about 15 min. Then was added hydroxyl-
amine hydrochloride (NH2OHꢁHCl) (neutralized by the NMM). The reaction mixture
was stirred until the completion of the reaction as indicated by TLC. After a simple
work up, the desired products were obtained in good yield. In this way several hydroxa-
mic acids (2a-2o) were synthesized from Na-protected Fmoc/Cbz/Boc amino acids

Hydroxamic Acids
3
R1
R1
BDMS, NMM
OH
NHOH
PgHN
PgHN
NH2OH.HCl,CH3CN
O
O
1
2
Pg = Fmoc/Boc or Cbz protecting group; R1 = CH3, CH(CH3)2, C6H5.
Scheme 1. Synthesis of Na-protected hydroxamic acids.
Table 1
List of Na-Protected Hydroxamic Acids Prepared following Scheme 1
25
Entry
Hydroxamic acids
Yield (%)
Mp (ꢀC)
[a]D in degrees
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
Na-Fmoc-Phe-NHOH
90
88
80
82
87
85
84
80
86
89
87
84
86
80
81
156
Gum
163
ꢂ0.012
ꢂ0.043
ꢂ0.008
ꢂ0.025
ꢂ0.023
ꢂ0.021
ꢂ0.029
ꢂ0.028
ꢂ0.018
ꢂ0.006
ꢂ0.020
ꢂ0.014
ꢂ0.079
ꢂ0.005
ꢂ0.015
a
ꢃ
N -Fmoc-Ala-NHOH
Na-Fmoc-Tyr-NHOH
Na-Fmoc-Pro-NHOH
Na-Fmoc-Val-NHOH
Na-Fmoc-Leu-NHOH
Na-Fmoc-Ile-NHOH
Na-Fmoc-Asp-NHOH
Na-Fmoc-Glu-NHOH
Na-Fmoc-Trp-NHOH
Na-Cbz-Phe-NHOH
Na-Cbz-Asp-NHOH
Na-Cbz-Trp-NHOH
Na-Boc-Phe-NHOH
Na-Boc-Tyr-NHOH
141
Gum
Gum
118
Gum
Gum
Gum
Gum
Gum
Gum
128
146
ꢃee for the product ¼ 97.67%.
1
(Table 1). The synthesized compounds were characterized by IR, H NMR, 13C NMR,
HRMS and elemental analysis.
The method was further extended for inserting the hydroxamic acid moiety into the
side chains of Asp/Glu. The key intermediates, N-protected Asp/Glu-5-oxazolidinones,
were prepared by treating N- protected amino acids with paraformaldehyde and a cata-
lytic amount of p-toluene sulfonic acid in toluene under reflux conditions. This reaction
was complete in 45 min as monitored by TLC. After the reaction, the mixture was diluted
with ethyl acetate and washed with water and brine solution. The organic layer was dried
over anhydrous Na2SO4 and finally concentrated. The resulting residue was recrystallized
using ethyl acetate and hexane. N-Protected Asp/Glu-5-oxazolidinones were treated with
NMM and BDMS at 0ꢀC. Then hydroxylamine hydrochloride was added. In the next
step, the oxazolidinone ring was selectively saponified with 1 N LiOH to obtain the free
a-COOH of Asp/Glu bearing hydroxamic acids in the side chains.
During the synthesis of the hydroxamates, the O-activation of hydroxamic acids
takes a longer time than that required for carboxy activation, so that an excess of
BDMS is required to drive the reaction towards completion. After the formation of the
isocyanates, we aimed to trap these isocyanates with nucleophiles such as amines and
alcohols. Ureas and carbamates were prepared starting from N-protected amino acids

4
Lalithamba et al.
R1
R1
R1
BDMS, NMM
Reflux Condition
BDMS, NMM
OH
NHOH
PgHN N C O
PgHN
PgHN
NH2OH.HCl,CH3CN
O
O
e
M
O
A
-
)
r
2
-
O
R
(
H
H
C
2
-
H
N
R1
O
R2
R1
O
X
PgHN
N
H
N
H
COOMe
PgHN
N
O
H
3
4
Pg = Fmoc/Boc or Cbz group;
R1, R2 = CH3, CH(CH3)2, CH2CH(CH3)2, CH2(C6H5); X=CH3, NO2.
Scheme 2. Synthesis of Na-protected ureas and carbamates.
Table 2
List of Na-Protected Ureas and Carbamates Prepared following Scheme 2
Entry
Na-protected Ureas and Carbamates
Yield (%)
Mp (ꢀC)
184
3a
85
O
OMe
FmocHN
FmocHN
FmocHN
N
H
N
H
C
O
3b
3c
4a
90
88
90
180
158
O
O
OMe
OMe
N
H
N
H
C
O
N
H
N
H
C
O
Gum
O
O
O
FmocHN
FmocHN
FmocHN
N
H
O
O
O
4b
4c
88
86
170
NO2
N
H
Gum
CH3
N
H

Hydroxamic Acids
5
along with BDMS and NMM in acetonitrile at 0ꢀC for 15 minutes, then NH2OHꢁHCl
was added. The reaction mixture was stirred until the reaction was completed. BDMS
and NMM were added again to the reaction mixture and it was subjected to reflux for
an hour. Then, the intermediate isocyanate was treated with the amino acid ester to give
the related ureidopeptide in good yields. The protocol was further extended to the syn-
thesis of carbamates, in which the intermediate isocyanate was trapped with an alcohol/
phenol and a simple workup led to isolation of pure ureas and carbamates (Scheme 2).
During this study, we also synthesized the set of enantiomeric N-Fmoc-L-Ala-
NHOH (a) and N-Fmoc-D-Ala-NHOH (b). The synthesis of hydroxamic acids employ-
ing BDMS was found to be racemization-free as indicated by the chiral-HPLC. A single
peak was observed at Rt 6.55 min for compound (a) and for its epimer (b) the Rt value
was at 7.38 min. This confirms that the BDMS mediated synthesis of Na-protected
hydroxamic acids is a racemization free protocol. Results are summarized in Table 2.
In silico Studies
To evaluate the binding efficacy and inhibitory effects of the synthesized compounds,
two protein targets from two different organisms, namely Escherichia coli in Figure 1a
and Staphylococcus aureus in Figure 1b, were selected for the study. The X-ray crystal-
lographic structure of enoyl-ACP reductase (1C14) E. coli33 and X-ray crystallographic
34
structure of dehydrosqualene synthase (2ZCP) S. aureus
respectively were obtained
from the Protein Data Bank (PDB). The prepared targets were then used for a molecular
docking study which predicts possible ligand interactions with the active site residues
that can inhibit protein activity.
To perform the docking, the FlexX module of LeadIT software was used35 and the
LeadIT scores obtained after the docking were considered to obtain the free binding
energy (DG). Based on the very low binding energy and maximum possible intermo-
lecular interactions, the best lead compounds with strong binding efficacy were deter-
mined. Two dimensional structures of all the ligands were drawn in ChemDraw Ultra
8.0 and were exported as mol files for further processing in DS 3.5. The generated con-
formers were optimized using the CHARMm force field36 and then minimized. The
selected conformers were grouped into one library file and were subjected to docking.
The FlexX docking score correlates with the binding affinity of the molecules with the
target. The docking scores of hydroxamic acids with E. coli and S. aureus are tabulated
in Table 3 (a) and Table 3 (b).
The ligand Fmoc-Tyr-NHOH showed a minimum binding energy of ꢂ14.78 kcal/
mol with E. coli and the ligand Fmoc-Trp-NHOH showed a minimum binding energy
of ꢂ15.07 kcal/mol with S. aureus. Figure 2 shows the pictorial representation of the
binding modes of the synthesized molecules with the target proteins. All the listed mol-
ecules in Table 3 (a) and Table 3 (b) were evaluated for in vitro studies. The com-
pounds Fmoc-Tyr-NHOH and Fmoc-Trp-NHOH showed significant antibacterial
activities, in accordance with the in silico results.
In vitro Antibacterial Activity
The antibacterial activities of the synthetic compounds was screened by an agar well dif-
fusion37 assay against the pathogenic bacterial strains E. coli [NCIM-5051] gram nega-
tive and S. aureus [NCIM-5022] gram positive. Preliminary screening was done to check
antibacterial activities of synthesized compounds over Mueller-Hinton agar plates.

6
Lalithamba et al.
Figure 1. a. Crystal structure of E. coli. b. Crystal structure of S aureus.
Table 3
(a) Docking Results of Hydroxamic Acids with E. coli (4V49)
Entry
Compound name
E. coli Docking score (kcal/mol)
1.
2.
3.
4.
5.
Fmoc-Tyr-NHOH
Fmoc-Ala-NHOH
Fmoc-Phe-NHOH
Cbz-Trp-NHOH
Boc-Phe-NHOH
ꢂ14.78
ꢂ14.42
ꢂ13.38
ꢂ8.630
ꢂ8.016
Table 3
(b) Docking Results of Hydroxamic Acids with S. aureus (1T2W)
Entry
Compound name
S. aureus Docking score (kcal/mol)
1.
2.
3.
4.
5.
Fmoc-Trp-NHOH
Fmoc-Asp-NHOH
Fmoc-Val-NHOH
Cbz-Asp-NHOH
Fmoc-Pro-NHOH
ꢂ15.07
ꢂ14.88
ꢂ14.81
ꢂ14.18
ꢂ9.990
In case of E. coli, the synthesized hydroxamic acids Fmoc-Tyr-NHOH and Fmoc-
Ala-NHOH showed a moderate zone of inhibition. The smallest zones of inhibition
were for Fmoc-Phe-NHOH, Cbz-Trp-NHOH and Boc-Phe-NHOH. S. aureus strains
exhibited significant inhibition for Fmoc-Trp-NHOH, Fmoc-Asp-NHOH, Fmoc-Val-
NHOH and the least inhibition for Cbz-Asp-NHOH and Fmoc-Pro-NHOH.
In sum, an efficient conversion of N-protected amino acids into hydroxamates
under reflux conditions using BDMS as carboxyl group activator was described. The
BDMS was employed to mediate the LR. The in situ generated isocyanates were

Hydroxamic Acids
7
Figure 2. Pictorial representations of the binding modes of synthesized molecules with tar-
get proteins.
utilized to synthesize urea and carbamate derivatives in one pot. Docking and antibac-
terial studies were reported.
Experimental Section
General
All reactants were purchased from Sigma-Aldrich and Merck and used without purifica-
tion. The solvents were distilled prior to use. Melting points were taken in open capilla-
ries and are uncorrected. TLC analysis was carried out using precoated silica gel F254
plates. IR spectra were recorded on Bruker Alpha-T FT-IR spectrometer (KBr windows,
2 cmꢂ1 resolution). 1H NMR and 13C NMR spectra were done on a Bruker AMX
400 MHz spectrometer using Me4Si as an internal standard and CDCl3 as a solvent.
Mass spectra were recorded on a Micromass Q-ToF Micro Mass Spectrometer. Optical

8
Lalithamba et al.
rotations were measured at 25ꢀC using Rudolf research analytical, Autopol-II,
Automatic Polarimeter. (Sodium-D-line 589 nm and C ¼ 1). The Pathogenic bacterial
strains were purchased from National Chemical Laboratory (NCL) Pune, India. We
have prepared 21 new compounds as noted below.
Synthesis of Hydroxamic Acids (2a-2o)
To a solution of N-protected amino acid (1 mmol) in acetonitrile (10 mL), at 0ꢀC, NMM
(1.5 mmol) and BDMS (1.2 mmol) were added. The reaction mixture was stirred at the
same temperature for 15 min, followed by the addition of NH2OHꢁHCl (1.2 mmol) neu-
tralized with NMM prior to the addition. The reaction mixture was stirred until the
reaction was complete. The mixture was diluted with EtOAc (15 mL), washed with
H2O (10 mL) and brine (10 mL). The organic phase was dried over Na2SO4, the solvent
was evaporated under reduced pressure to yield the product.
Ureidopeptide and Carbamate Synthesis (3a-3c and 4a-4c)
To a chilled (0ꢀC) solution of N-protected amino acid (1 mmol) in acetonitrile (10 mL),
NMM (1.5 mmol) and BDMS (1.2 mmol) were added. The reaction mixture was stirred
at the same temperature for 15 min, followed by the addition of NH2OHꢁHCl
(1.2 mmol) neutralized with NMM prior to the addition. The reaction mixture was
stirred for 60 minutes. BDMS (1.2 mmol) and NMM (1.5 mmol) were added to the reac-
tion mixture and subjected to reflux for an hour. Then an amine or alcohol were added
(1.2 mmol) to give urea or carbamate product respectively. Then, the solvent was
removed under reduced pressure, the residue was diluted with EtOAc (15 mL) and the
organic layer was washed with 10% HCl (10 mL), Na2CO3 (10 mL), H2O (10 mL) and
brine (10 mL), then dried over anhydrous Na2SO4. The solvent was evaporated to afford
the product.
Na-Fmoc-Phe-NHOH: (2a) Yield 90%; Solid; Melting Point 156ꢀC; IR: (KBr, cmꢂ1
)
1
1640; H NMR (400 MHz, CDCl3) d (ppm) 2.08 (s, 1H), 2.91–3.12 (m, 2H), 4.40 (t, J
8.0 Hz, 1H), 4.75 (d, J 4.0 Hz, 2H), 4.98 (t, J 8.0 Hz, 1H), 5.05 (s, 2H), 7.10–7.77 (m,
13H). 13C NMR (100 MHz, CDCl3) d (ppm) 37.0, 46.5, 51.25, 66.55, 126.0, 126.80,
127.80, 128.20, 128.40, 128.70, 128.80, 139.5, 141.0, 143.6, 156.0, 169.5. MS: Cald.
for C24H22N2O4 m/z: 402.1578 (Mþ þNa), Found: 402.1576.
Anal. Calcd for C24H22N2O4: C, 71.63; H, 5.51; N, 6.96. Found: C, 71.51; H, 5.45;
N, 6.89.
Na-Fmoc-Ala-NHOH: (2b) Yield 84%, Gum. IR (KBr): 1637 cmꢂ1
.
1H NMR
(400 MHz, CDCl3): d (ppm) 1.36 (d, J 8.0 Hz, 3H), 1.99 (s, 1H), 4.22 (m, 1H), 4.41 (t,
J 8.0 Hz, 2H), 5.0 (s, 2H), 5.36 (s, 1H), 7.26-7.77 (m, 8H). 13C NMR (100 MHz,
CDCl3): d (ppm) 18.73, 47.13, 47.20, 67.04, 120.01, 125.10, 127.09, 127.73, 141.35,
141.82, 156.0, 169.50. MS: Cald. for C18H18N204 m/z: 326.1267 (Mþ þNa),
Found: 326.1265.
Anal. Calcd for C18H18N204: C, 66.25; H, 5.56; N, 8.58. Found: C, 66.32; H, 5.51;
N, 8.63.
Na-Fmoc-Tyr-NHOH: (2c) Yield 80%, Solid, Melting Point 163ꢀC. IR (KBr):
1
1651 cmꢂ1. H NMR (400 MHz, CDCl3): d (ppm) 1.75 (s, 2H), 2.81 (m, 2H), 4.27 (t, J

Hydroxamic Acids
9
8.0 Hz, 1H), 4.85 (s, 2H), 6.0 (s, 1H), 6.10 (s, 2H), 6.80–7.10 (m, 4H), 7.30–7.85 (m,
8H). 13C NMR (100 MHz, CDCl3): d (ppm) 37.52, 47.0, 51.25, 67.85, 119.82, 126.80,
128.20, 128.45, 128.69, 129.20, 141.0, 143.60, 155.70, 156.68, 169.50. MS: Cald. for
C24H22N2O5 m/z: 418.1529 (Mþ þNa), Found: 418.1531.
Anal. Calcd for C24H22N2O5: C, 68.89; H, 5.30; N, 6.69. Found: C, 68.78; H, 5.27;
N, 6.53.
Na-Fmoc-Pro-NHOH: (2d) Yield 82%, Solid, Melting Point 141ꢀC. IR (KBr):
1
1660 cmꢂ1. H NMR (400 MHz, CDCl3): d (ppm) 0.9 (m, 2H), 1.30 (m, 2H), 1.92 (s,
1H), 2.75 (m, 2H), 4.27 (t, J 4.0 Hz, 1H), 4.50 (br, 1H), 4.70 (d, J 8.0 Hz, 2H), 5.05 (s,
1H), 7.30–7.79 (m, 8H). 13C NMR (100 MHz, CDCl3): d (ppm) 22.0, 28.56, 47.0,
48.15, 48.66, 66.90, MS: Cald. for C20H20N2O4 m/z: 352.1423 (MþþNa),
Found: 352.1420.
Anal. Calcd for C20H20N2O4: C, 68.17; H, 5.72; N, 7.95. Found: C, 68.09; H, 5.61;
N, 7.83.
Na-Fmoc-Val-NHOH: (2e) Yield 87%, Gum. IR (KBr): 1635 cmꢂ1
.
1H NMR
(400 MHz, CDCl3): d (ppm) 0.97-0.99 (d, J 8.0 Hz, 6H), 2.0 (s, 1H), 2.16 (m, 1H), 4.28
(m, 1H), 4.35 (m, 1H), 4.43 (d, J 4.0 Hz, 2H), 5.30 (br, 2H), 7.26–7.77 (m, 8H). 13C
NMR (100 MHz, CDCl3): d (ppm) 17.63, 31.31, 47.22, 59.04, 67.04, 115.98, 125.0,
127.06, 127.70, 141.32, 143.79, 156.0, 168.58. MS: Cald. for C20H22N2O4 m/z:
354.1580 (MþþNa), Found: 354.1582.
Anal. Calcd for C20H20N2O4: C, 67.78; H, 6.26; N, 7.90. Found: C, 67.70; H, 6.22;
N, 8.02.
Na-Fmoc-Leu-NHOH: (2f) Yield 85%, Gum. IR (KBr): 1642 cmꢂ1
.
1H NMR
(400 MHz, CDCl3): d (ppm) 0.94 (d, J 4.0 Hz, 6H), 1.1–1.40 (m, 3H), 1.51–1.80 (m,
1H), 2.03 (s, 1H), 4.10–4.20 (m, 1H), 4.23-4.39 (t, J 8.0 Hz, 1H), 4.58 (d, J 4.0 Hz,
2H), 5.98–6.0 (t, J 8.0 Hz, 1H), 7.24–7.75 (m, 8H). 13C NMR (100 MHz, CDCl3): d
(ppm) 21.83, 22.78, 41.69, 47.16, 52.44, 66.92, 120.07, 126.46, 127.64, 130.01, 141.40,
143.76, 155.97, 173.67. MS: Cald. for C21H24N2O4 m/z: 368.1736 (MþþNa),
Found: 368.1732.
Anal. Calcd for C21H24N2O4: C, 68.46; H, 6.57; N, 7.60. Found: C, 68.37; H, 6.51;
N, 7.51.
Na-Fmoc-Ile-NHOH: (2g) Yield 84%, Solid, Melting Point 118ꢀC. IR (KBr):
1
1638 cmꢂ1. H NMR (400 MHz, CDCl3): d (ppm) 0.92 (br, 3H), 1.25 (s, 3H), 1.61 (br,
2H), 1.92 (s, 1H), 2.04 (m, 1H), 4.32 (m, 1H), 4.40 (m, 1H), 4.60 (s, 2H), 5.28 (br,
2H), 7.29–7.77 (m, 8H). 13C NMR (100 MHz, CDCl3): d (ppm) 10.90, 14.65, 24.70,
36.84, 47.75, 53.86, 67.40, 127.11, 128.20, 128.35, 128.86, 141.0, 143.66, 156.0,
168.69. MS: Cald. for C21H24N2O4 m/z: 368.1736 (Mþ þNa), Found: 368.1730.
Anal. Calcd for C21H24N2O4: C, 68.46; H, 6.57; N, 7.60. Found: C, 68.40; H, 6.49;
N, 7.64.
Na-Fmoc-Asp-NHOH: (2h) Yield 80%, Gum. IR (KBr): 1648 cmꢂ1 1H NMR
.
(400 MHz, CDCl3): d (ppm) 1.98 (s, 1H), 4.43 (t, J 12.0 Hz, 1H), 4.48 (d, J 8.0 Hz,
2H), 5.0 (s, 1H), 5.65 (s, 2H), 6.0 (s, 1H), 7.28–7.8 (m, 8H). 13C NMR (100 MHz,
CDCl3): d (ppm) 45.6, 66.7, 68.5, 77.2, 126.4, 128.0, 128.4, 128.6, 140.0, 142.8, 152.8,
166.0, 170.9. MS: Cald. for C19H16N2O6 m/z: 391.100 (MþþNa), Found: 391.1002.
Anal. Calcd for C19H16N2O6: C, 61.95; H, 4.38; N, 7.61. Found: C, 61.90; H, 4.29;
N, 7.54.

10
Lalithamba et al.
Na-Fmoc-Glu-NHOH: (2i) Yield 86%, Gum. IR (KBr): 1690 cmꢂ1
.
1H NMR
(400 MHz, CDCl3): d (ppm) 1.85 (s, 1H), 2.60 (d, J 8.0 Hz, 2H), 4.4 (t, J 12.0 Hz, 1H),
4.6 (t, J 12.0 Hz, 1H), 4.7 (d, J 8.0 Hz, 2H), 5.67 (s, 2H), 6.0 (s, 1H), 7.2–7.75 (m, 8H).
13C NMR (100 MHz, CDCl3): d (ppm) 26.5, 46.8, 53.7, 66.9, 77.4, 126.0, 127.9, 128.2,
128.8, 141.0, 143.4, 154.7, 170.6, 174.5. MS: Cald. for C20H18N2O6 m/z: 405.12
(MþþNa), Found: 405.1180.
Anal. Calcd for C20H18N2O6: C, 62.82; H, 4.74; N, 7.33. Found: C, 62.73; H, 4.68;
N, 7.44.
Na-Fmoc-Trp-NHOH: (2j) Yield 89%, Gum. IR (KBr): 1656 cmꢂ1
.
1H NMR
(400 MHz, CDCl3): d (ppm) 2.04 (s, 1H), 3.25 (d, J 8.0 Hz, 2H), 4.35 (s, 1H), 4.42 (d,
2H), 4.90 (t, 1H), 5.00 (s, 3H), 6.09 (s, 1H), 7.26–7.71 (m, 12H). 13C NMR (100 MHz,
CDCl3): d (ppm) 31.24, 47.65, 52.15, 67.46, 110.9, 111.35, 118.86, 120.28, 122.20,
122.76, 126.80, 127.45, 128.20, 128.45, 128.80, 136.50, 141.0, 143.68, 156.0, 168.78.
MS: Cald. for C26H23N3O4 m/z: 441.1689 (MþþNa), Found: 441.1688.
Anal. Calcd for C26H23N3O4: C, 70.73; H, 5.25; N, 9.52. Found: C, 70.68; H, 5.16;
N, 9.45.
1
Na-Cbz-Phe-NHOH: (2k) Yield 82%, Gum. IR (KBr): 1660 cmꢂ1. H NMR (400 MHz,
CDCl3): d (ppm) 2.78 (s, 1H), 3.24 (m, 2H), 4.38 (m, 1H), 5.24 (s, 2H), 5.50 (br, 2H),
7.24–7.51 (m, 10H). 13C NMR (100 MHz, CDCl3): d (ppm) 37.89, 52.36, 67.03,
127.17, 128.22, 128.65, 129.30, 135.80, 136.29, 155.78, 159.37, 172.73, 173.40. MS:
Cald. for C17H18N2O4 m/z: 314.1267 (MþþNa), Found: 314.1269.
Anal. Calcd for C17H18N2O4: C, 64.96; H, 5.77; N, 8.91. Found: C, 65.03; H, 5.64;
N, 8.82.
1
Na-Cbz-Asp-NHOH: (2l) Yield 84%, Gum. IR (KBr): 1655 cmꢂ1. H NMR (400 MHz,
CDCl3): d (ppm) 2.0 (s, 1H), 5.3 (s, 2H), 5.38 (s, 1H), 5.64 (s, 2H), 6.0 (s, 1H), 7.18 (s,
5H). 13C NMR (100 MHz, CDCl3): d (ppm) 65.6, 68.4, 77.2, 127.4, 127.6, 128.9,
154.4, 166.0, 170.8. MS: Cald. for C12H12N2O6 m/z: 303.07 (MþþNa),
Found: 303.0670.
Anal. Calcd for C17H18N2O4: C, 51.43; H, 4.32; N, 10.00. Found: C, 51.32; H,
4.24; N, 10.08.
Na-Cbz-Trp-NHOH: (2m) Yield 86%, Gum. IR (KBr): 1658 cmꢂ1
.
1H NMR
(400 MHz, CDCl3): d (ppm) 2.14 (s, 1H), 3.04 (s, 2H), 4.26 (s, 1H), 4.89 (m, 2H), 4.99
(s, 3H), 6.82–7.93 (m, 10H). 13C NMR (100 MHz, CDCl3): d (ppm) 31.20, 50.27,
65.90, 110.86, 111.08, 119.26, 120.0, 122.15, 122.90, 127.25, 127.56, 127.80, 129.05,
136.43, 141.28, 156.98, 168.70. MS: Cald. for C19H19N3O4 m/z: 353.2176 (MþþNa),
Found: 353.2174.
Anal. Calcd for C19H19N3O4: C, 64.58; H, 5.42; N, 11.89. Found: C, 64.46; H,
5.37; N, 11.77.
Na-Boc-Phe-NHOH: (2n) Yield 80%, Solid, Melting Point 128ꢀC. IR (KBr):
1
1639 cmꢂ1. H NMR (400 MHz, CDCl3): d (ppm) 1.39 (s, 9H), 2.27 (s, 1H), 3.09 (m,
2H), 4.33 (m, 1H), 4.95 (br, 2H), 7.18–7.30 (m, 5H). 13C NMR (100 MHz, CDCl3): d
(ppm) 28.25, 37.50, 51.17, 72.55, 126.0, 127.85, 128.69, 139.44, 156.0, 169.48. MS:
Cald. for C14H20N2O4 m/z: 280.1423 (MþþNa), Found: 280.1422.
Anal. Calcd for C14H20N2O4: C, 59.99; H, 7.19; N, 9.99. Found: C, 59.87; H, 7.11;
N, 9.87.

Hydroxamic Acids
11
Na-Boc-Tyr-NHOH: (2o) Yield 81%, Solid, Melting Point 146ꢀC. IR (KBr):
1
1643 cmꢂ1. H NMR (400 MHz, CDCl3): d (ppm) 1.41-1.60 (m, 9H), 1.76 (m, 2H),
3.14 (d, J 4.0 Hz, 2H), 4.34 (m, 1H), 5.00 (s, 2H), 6.75–6.81 (m, 4H). 13C NMR
(100 MHz, CDCl3): d (ppm) 28.27, 37.56, 64.91, 79.50, 115.51, 121.33, 130.42, 155.0,
155.68, 169.43. MS: Cald. for C14H20N2O5 m/z: 296.1372 (MþþNa), Found: 296.1368.
Anal. Calcd for C14H20N2O5: C, 56.75; H, 6.80; N, 9.45. Found: C, 56.67; H, 6.73;
N, 9.34.
Na-Fmoc-Phe-w[NHCONH]-Ala-OMe: (3a) Yield 83%; Solid; Melting Point 185ꢀC;
1
IR: (KBr, cmꢂ1) 3355; H NMR (400 MHz, DMSO-d6): d (ppm) 0.83 (s, 3H), 3.40 (m,
2H,), 3.39 (s, 3H), 4.32 (m, 1H), 4.58 (d, J 6.6 Hz, 1H), 4.75 (d, J 10.0 Hz, 2H, 5.02
(br, 3H), 5.82 (t, J 8.0 Hz,1H), 7.20–7.85 (m, 13H). 13C NMR (100 MHz, DMSO-d6): d
(ppm) 20.05, 38.0, 46.54, 48.9, 51.86, 62.12, 66.30, 120.55, 125.54, 126.90, 127.51,
128.2, 128.81,129.53, 138.42, 141.56, 144.51, 156.8, 157.40,174.0. MS: Cald. for
C28H29N3O5 m/z: 510.2005 (M þ Naþ), found 510.2005.
Anal. Calcd for C28H29N3O5: C, 68.98; H, 6.00; N, 8.62. Found: C, 68.90; H, 5.93;
N, 8.56.
Na-Fmoc-Ala-w[NHCONH]-Val-OMe: (3b) Yield 90%; Solid; Melting Point 182ꢀC;
1
IR: (KBr, cmꢂ1) 3288; H NMR (400 MHz, DMSO-d6): d (ppm) 1.01 (br, 6H), 1.16-
1.94 (br, 3H), 3.05 (m. 1H), 3.55 (s, 3H), 4.0 (d, J 12.0 Hz, 1H), 4.15–4.24 (br, 1H),
4.51–4.82 (br, 2H), 5.13 (br, 1H), 6.31-6.37 (br, 3H), 7.22-7.94 (m, 8H). 13C NMR
(100 MHz, DMSO-d6): d (ppm) 18.34, 20.75, 31.28, 45.18, 51.35, 53.82, 59.74, 66.15,
126.53, 126.81, 127.12, 128.39, 141.12, 144.23, 156.02, 157.23, 174.12. MS: Cald. for
C24H29N3O5 m/z: 462.2005 (M þ Naþ), found 462.2008.
Anal. Calcd for C24H29N3O5: C, 65.59; H, 6.65; N, 9.56. Found: C, 65.50; H, 6.76;
N, 9.63.
Na-Fmoc-Leu-w[NHCONH]-Ala-OMe: (3c) Yield 86%; Solid, Melting Point 161ꢀC;
1
IR: (KBr, cmꢂ1) 3271; H NMR (400 MHz, DMSO-d6): d (ppm) 0.96 (t, J 10.0 Hz,
3H), 1.10 (d, J 6.0 Hz, 6H), 1.22 (d, J 10.0 Hz, 3H), 1.38 (m, 2H), 2.90 (m, 1H), 3.14
(m, 1H), 3.65 (s, 3H), 4.09 (t, J 8.0 Hz, 1H), 4.45 (t, J 12.0 Hz, 1H), 4.80 (d, J 8.0 Hz,
2H), 5.66 (m, 1H), 6.0 (br, 3H), 7.25–7.85 (m, 8H). 13C NMR (100 MHz, DMSO-d6): d
(ppm) 11.65, 12.35, 17.20, 22.48, 30.13, 38.8, 47.3, 51.7, 57.6, 65.9, 67.8, 126.8, 128.4,
128.6, 128.8, 140.9, 143.8, 156.2, 157.9, 170.8. MS: Calc. for C27H35N3O5 m/z: 504.25
(M þ Naþ), found 504.2214.
Anal. Calcd for C27H35N3O5: C, 67.34; H, 7.33; N, 8.73. Found: C, 67.46; H, 7.14;
N, 8.67.
Na-Fmoc-Phe-w[NHCO]-OC6H5: (4a) Yield 90%, Gum, IR (KBr): 1638 cmꢂ1 1H
.
NMR (400 MHz, DMSO-d6): d (ppm) 2.8 (t, J 8.0 Hz, 2H), 4.45 (m, 1H), 4.59 (d, J
6.0 Hz, 2H), 6.0 (s, 3H), 7.03–7.8 (m, 18H). 13C NMR (100 MHz, DMSO-d6): d (ppm)
42.2, 45.0, 63.23, 67.38, 121.4, 125.6, 126.0, 126.65, 127.46, 128.2, 128.38, 128.7,
128.85, 129.2, 139.0, 141.0, 143.7, 151.5, 155.8. MS: Cald. for C30H26N2O4 m/z:
501.19 (MþþNa), Found: 501.1890.
Anal. Calcd for C30H26N2O4: C, 75.30; H, 5.48; N, 5.85. Found: C, 75.40; H, 5.59;
N, 5.76.
Na-Fmoc-Phe-w[NHCO]-OC6H5NO2: (4b) Yield 88%, Solid, Melting Point 170ꢀC IR
1
(KBr): 1656 cmꢂ1. H NMR (400 MHz, DMSO-d6): d (ppm) 2.95 (t, J 8.0 Hz, 2H),
4.38 (t, J 12.0 Hz, 1H), 4.79 (d, J 6.0 Hz, 2H), 6.0 (s, 5H), 7.1–7.84 (m, 15H). 13C

12
Lalithamba et al.
Table 4
Antibacterial Test for Protected Hydroxamic Acids with E. coli & S. aureus
Treatment
Treatment
(Concentration E. coli
in mg/mL) (Mean SE) Sample
(Concentration S. aureus
in mg/mL) (Mean SE)
Sample
Ciprofloxacin
5
500
1000
500
1000
500
1000
500
1000
500
10.13 0.06 Ciprofloxacin
1.53 0.03 Fmoc-Trp-
3.37 0.07 NHOH
1.03 0.03 Fmoc-Asp-
2.40 0.06 NHOH
1.07 0.03 Fmoc-Val-
2.03 0.03 NHOH
0.53 0.03 Cbz-Asp-
1.23 0.03 NHOH
5
500
1000
500
1000
500
1000
500
1000
500
11.13 0.03
3.17 0.03
5.90 0.06
2.00 0.03
3.60 0.06
1.40 0.06
3.13 0.03
Inactive
Fmoc-Tyr-NHOH
Fmoc-Ala-NHOH
Fmoc-Phe-NHOH
Cbz-Trp-NHOH
Boc-Phe-NHOH
2.17 0.09
1.23 0.12
2.13 0.09
In active Fmoc-Pro-
1.10 0.06 NHOH
1000
1000
NMR (100 MHz, DMSO-d6): d (ppm) 41.5, 47.0, 63.4, 66.8, 121.5, 122.8, 126.0, 126.5
126.8, 127.4, 128.2, 128.4, 128.8, 129.0, 139.5, 141.0, 143.4, 145.2, 154.6, 156.0,
157.5. MS: Cald. for C30H25N3O6 m/z: 546.17 (MþþNa), Found: 546.1681
C, H, N
Anal. Calcd for C30H25N3O6: C, 68.82; H, 4.81; N, 8.03. Found: C, 68.93; H, 4.90;
N, 7.94.
1
Na-Fmoc-Phe-w[NHCO]-OC6H5CH3: (4c) Yield 86%, Gum, IR (KBr): 1660 cmꢂ1. H
NMR (400 MHz, DMSO-d6): d (ppm) 2.36 (d, J 6.0 Hz, 3H), 2.9 (d, J 6.0 Hz, 2H), 4.38
(t, J 8.0 Hz, 1H) 4.66 (d, J 6.0 Hz, 2H), 6.0 (s, 3H), 6.95–7.84 (m, 17H). 13C NMR
(100 MHz, DMSO-d6): d (ppm) 24.5, 42.2, 46.8, 63.4, 67.6, 121.5, 126.0, 126.5, 127.8,
128.0, 128.2, 128.6, 128.8, 129.5, 135.0, 139.8 141.1, 143.5, 148.2, 154.6, 155.8. MS:
Cald. for C31H28N2O4 m/z: 515.2 (MþþNa), Found: 515.2001.
Anal. Calcd for C31H28N2O4: C, 75.59; H, 5.73; N, 5.69. Found: C, 75.64; H, 5.85;
N, 5.60.
In silico Studies
The purpose of the study was to analyze the inhibitory action of the synthesized hydroxa-
mic acids by computational docking. Such in silico methods are extremely useful to find
out potential binding sites and also to discover new molecules that could bind to a known
site. In this study, the target protein and hydroxamic acid molecule were optimized by
drug discovery studio version 3.5 software available from Accelrys Discovery Studio.
In vitro Antibacterial Activity
Agar plates were inoculated with a standardized inoculum of the test microorganism.
Then, the test compound at a desired concentration was placed on the agar surface and
petri dishes were incubated under appropriate conditions. Generally, the antimicrobial

Hydroxamic Acids
13
agent diffuses into the agar and inhibits the growth of test microorganism and then the
diameters of inhibition growth zones are measured.
The nutrient medium was prepared by dissolving 37.0 g of nutrient agar medium in
1000 ml of distilled water and adjusting pH to 7.3 ( 0.1), subjecting it to sterilization in
an autoclave at 121ꢀC for 15–30 min. Nutrient agar plates were prepared and 20–25 ml
of sterile nutrient agar medium was poured into sterile petri-dishes and allowed to solid-
ify. Then, 100 ml of mature broth culture of individual pathogenic bacterial strains was
spread all over the surface of agar plates using sterilized L-shaped glass rod. About
6 mm well are made in each nutrient agar plate using sterile cork borer. Different con-
centrations of synthetic compounds were used to assess the dose dependent activity of
the product. Antibacterial activity for listed molecules in Table 3 (a) and Table 3 (b)
were carried out. At two different concentrations 500 mg/mL and 1000 mg/mL of synthe-
sized molecules, the inhibition zones were measured using vernier callipers in mm and
its values were depicted in Table 4. Simultaneously the standard antibiotic
(Ciprofloxacin used as a positive control) was tested against the pathogenic bacterial
strains. Then, the plates were incubated at 37ꢀC for 36 h. After incubation, the zone of
inhibition of each well was measured and the values were recorded. The experiments
were carried out in triplicates with each compound and the average values were calcu-
lated for determining the antibacterial activity.
Acknowledgments
We thank the Principal and Director of Siddaganga Institute of Technology,
Tumakuru, Karnataka, for the research facilities. One of the authors (HSL) is thankful
to the Vision Group of Science and Technology, Department of Information
Technology, Biotechnology and Science & Technology, Government of Karnataka, for
providing funds under the CISEE Programme (GRD No. 472) to carry out the present
research work by means of a sponsored project.
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