Synthesis of (2-mercaptoacetyl)-L-[2-14C]tryptophan as a selective metallo-β-lactamase inhibitor via [2-14C]indole based on chiral pool strategy

Article abstract of DOI:10.1002/jlcr.3485

Metallo-beta-lactamase enzymes make bacteria resistant to a broad range of commonly used beta-lactam antibiotics. Several thiol derivatives of L-amino acids have been shown their inhibitory effects against the metallo-β-lactamase IMP-1. In this study, (2-mercaptoacetyl)-L-tryptophan as a new inhibitor of metallo-β-lactamases labeled with carbon-14 in the 2-position of the indole ring was prepared from [2-¹⁴C]indole as a key synthetic intermediate based on chiral pool strategy. The overall synthesis was performed in 10 steps with the overall radiochemical yield 3.6% on the basis of the barium [¹⁴C]carbonate as a starting material.

Full text of DOI:10.1002/jlcr.3485

Synthesis of (2-mercaptoacetyl)-L-[2-14C]tryptophan as a selective metallo-
-lactamase inhibitor via [2-14C]indole based on chiral pool strategy
Gholamhossein Shirvania, Abbas Shockravib*, Mohsen Aminic, Nader Saemiana§
a
Nuclear Science Research School, Nuclear Science & Technology Research Institute, End of North
Karegar Ave, Postal Code: 14395-836, Tehran, Iran.
b*
Correspondence to: Abbas Shockravi, Faculty of Chemistry, Kharazmi University, 49 Mofetteh Avenue,
Postal Code: 15719-14911, Tehran, Iran. E-mail: abbas_shockravi@yahoo.co.uk, Tel: +982188329220-
3, Cell Phone: +989122596174, Fax: +982188825580
c
Department of Medicinal Chemistry, Faculty of Pharmacy and Drug Design Development Research
Center, Tehran University of Medical Sciences, Tehran 14176, Iran
§This paper is dedicated to memory of Dr. Nader Saemian.
Abstract
Metallo-beta-lactamase enzymes make bacteria resistant to a broad range of commonly
used beta-lactam antibiotics. Several thiol derivatives of L-amino acids have been shown
their inhibitory effects against the metallo--lactamase IMP-1. In this study, (2-
mercaptoacetyl)-L-tryptophan as a new inhibitor of MBLs labeled with carbon-14 in the 2-
position of the indole ring was prepared from [2-14C]indole as a key synthetic intermediate
based on chiral pool strategy. The overall synthesis was carried out in ten-step with the
overall radiochemical yield 3.6% based on the barium [14C]carbonate as a starting material.
Keywords: Antibiotics; Indole; MBL’s inhibitors; Carbon-14; Amino acid
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doi: 10.1002/jlcr.3485
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Introduction
Beta-lactam antibiotics were introduced for many years and account for more than 50
percent of prescription drugs.1 This type of antibiotics is one of the most important branches
of antibiotics including carbapenems, cephalosporins, penicillins, and monobactams (Figure
1).2-5 A major problem in chemotherapy of infection diseases is the development of resistance
against anti-bacterial agents.6 Different mechanisms were suggested for the development of
resistance in microorganisms. One of the important methods is secretion of such enzymes that
could deactivate antibiotics. This class of enzyme is known as β-lactamase, which includes
serine β-lactamases (SBLs, Classes A,C and D) and metallo-β-lactamase (MBLs, Class B)7
which open the β-lactam ring and inactivate them.8, 9 Inactivation of antibiotics causes the
reactivation of the disease and is considered a serious threat to human health.10, 11 For
instance, amoxicillin, an antibiotic that is clinically unable to cope with this type of enzymes.
Accordingly, new inhibitors of enzymes are of an urgent and fundamental need.
Various classes of MBL inhibitors have been reported, including dicarboxylic acids,
thiols, sulfates, hydroxamates, tetrazoles, trifluoromethyl ketones, alcohols, and
sulfonamides. 12-30 N-3-mercapto propanoyl derivative of unnatural D-phenylalanine has been
reported as a competitive Inhibitor of Metallo-β-lactamase (IMPs).
The synthesis and antibacterial evaluation of thiol derivatives for three natural
aromatic amino acid, L-tryptophan, L-tyrosine and phenyl alanine showed good antibacterial
activity and introduced a new class of the chemical structure as potent antimicrobial agent.
All of the compounds in Figure 2 show the inhibitory properties against IMP-1. One of them,
compound d (Figure 2), (2-Mercaptoacetyl)-L-[2-14C]tryptophan (n=1), showed a very
interesting potency as antibiotic.31
(2-Mercaptoacetyl)-L-tryptophan was synthesized based on the docking studies and
used in kinetic enzyme assays and cellbased assays.31 In order to further clarify mechanism of
action and specify pharmacological interaction, synthesis of 14C-labeled (2-mercaptoacetyl)-
L-tryptophan was required.. In this paper, we report continuation of our previous work on the
synthesis of 14C-labeled inhibitors,32 and, specifically, a convenient synthetic pathway for
the preparation of compound d, (n=1) in which 2-position of indole ring was labeled with
carbon-14 (Scheme 1).
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Results and discussion
Formic [14C]acid 3 was prepared from barium [14C]carbonate 2 under standard
procedure by reduction of 14CO2 by LiEt3BH.33 N-[14C]formyl-o-toluidine 5 was derived from
reaction of o-toluidine 4 and formic [14C]acid 3.34 Compound 5 was converted to 1H-[2-
14C]indole 1 under heating and in the presence of strong base.35
1-(Tert-butyldimethylsilyl)-1H-[2-14C]indole 6 was prepared by reaction between 1H-
[2-14C]indole and TBDMSCl in good yield. The conversion of compound 6 to 3-bromo-1-
(tert-butyldimethylsilyl)-1H-[2-14C]indole 7 was carried out from reaction of NBS with 6 in
THF at low temperature. N-(tert-butoxycarbonyl)-1-(tert-butyldimethylsilyl)-L-[2-
14C]tryptophan methyl ester 9 was produced by reaction between 7 and Iodo-Boc-Ala-OMe
8 in the presence of tert-butyl lithium.35 The TBDMS protecting group was removed and
compound 10 was obtained,36 and deprotection of later compound by TFA in DCM led to L-
[2-14C]tryptophan methyl ester 11 in good yield.37-42 Acylation of 11 was accomplished by
HBTU and DIPEA with standard procedure and 12 was obtained.43 Hydrolysis of 12 was
carried out in one step and the titled compound 13 was obtained.31
Experimental
1H NMR spectra were recorded on Bruker AVANCE 500 spectrometer.
Formic [14C]acid (3)
Barium [14C]carbonate (24 mmol, 20 MBq/mmol, 480.3 MBq) was converted to formic
[14C]acid (18.37 mmol, 367.4 MBq) in the presence LiBEt3H (36.6 mL, 1 mol/L) in THF
according to known procedure in 76.5% radiochemical yield and product was collected for
the next step.33
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N-[14C]formyl-o-toluidine (5)
Formic [14C]acid (18.37 mmol, 367.4 MBq) and o-toluidine (19.33 mmol, 2.07 g) was
mixed and the mixture kept under reflux conditions on a water bath for 4 h. The reaction
mixture was stand in refrigerator overnight and the N-[14C]formyl-o-toluidine was solidified.
The solid product was filtered and washed with 0.1M HCl (2×10 mL) and then water (2×10
mL), dried in vacuo in desiccator over P2O5. The solid was recrystallized from benzene-light
petroleum (1:2, v/v) to give N-[14C]formyl-o-toluidine (13.65 mmol, 1.87 g, 273 MBq) in
74% radiochemical yield. 1H NMR (CDCl3) (ppm): 8.55 (s, 1H), 8.42 (br, s, 1H, NH), 7.26-
7.06 (m, 4H), 2.31 (s, 3H).
1H-[2-14C]indole (1)
N-[14C]formyl-o-toluidine (13.65 mmol, 1.87 g, 273 MBq), t-BuOK (25.81 mmol, 2.90
g) and t-BuOH (4.4 mL) was mixed and stirred for 10 min. Then excess t-BuOH distilled off
and the residue heated gently to 350-360 ºC for 15 minutes. The mixture was cooled and
ethyl acetate was added to the mixture and the organic layer was separated, dried over sodium
sulfate and the residue was purified by silica gel chromatography using ethyl acetate:n-
hexane (30-70%) as eluent to give the titled compound 1 (3.71 mmol, 0.44 g, 74.3 MBq) in
27.2% radiochemical yield. 1H NMR (CDCl3) (ppm): 8.05 (1H, bs, NH), 7.64 (1H, d), 7.37
(1H, d), 7.22-7.10 (3H, m), 6.68 (1H, bd).
3-Bromo-1-(tert-butyldimethylsilyl)-1H-[2-14C]indole (7)
A solution of n-butyllithium in n-hexane (3 mL, 1.6 M solution) was added dropwise to
a solution of 1 (3.71 mmol, 0.44 g, 74.3 MBq) in 15 mL of anhydrous THF at -78 ºC and the
temperature was raised to -10 ºC and stand at this temperature for 15 minutes. Then reaction
mixture was cooled to -50 ºC, a solution of tert-butyldimethylsilyl chloride (4.15 mmol,
dissolved in 3.3 mL of anhydrous THF) was added and then mixture temperature was raised
to 0 ºC and was stirred for 3 hours. After that, the temperature was lowered to -78 ºC, and
freshly crystallized N-bromosuccinimide (3.71 mmol, 0.66 g) was added to the reaction
mixture. The reaction mixture was stirred for two hours and then the temperature was raised
to 25 ºC, and n-hexane (11 mL) and pyridine (0.1 mL) were added. The reaction mixture was
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filtered and the filtrate was evaporated in vacuo. The crude mixture was purified by silica gel
chromatography using ethyl acetate:n-hexane (20-80%) as eluent to give the titled compound
1
7 (3.00 mmol, 0.93 g, 60.1 MBq) in 81% radiochemical yield. H NMR (CDCl3) (ppm):
7.57-7.55 (1H, m), 7.48-7.46 (1H, m), 7.20-7.16 (3H, m), 0.91 (9H, s), 0.60 (6H, s).
N-(tert-butoxycarbonyl)-1-(tert-butyldimethylsilyl)-L-[2-14C]tryptophan
methyl ester (9)
To the solution of 7 (3.00 mmol, 0.93 g, 60.1 MBq) in anhydrous THF (19 mL) was
added at -78 C a solution of tert-butyllithium in pentane (6.77 mmol, 4.6 mL, 1.48 M
solution) and was stirred at this temperature for 20 min. Then Iodo-Boc-ala-OMe 8 (6.00
mmol, 1.98 g) dissolved in THF (10 mL) at -78 C, was poured into the reaction mixture and
stirred for 5 hours. Then, the reaction solution was evaporated to dryness and the crude
mixture was purified by silica gel chromatography using ethyl acetate:n-hexane (30-70%) as
eluent to give the titled compound 9 (2.04 mmol, 0.89 g, 40.9 MBq) in 68% radiochemical
1
yield. H NMR (CDCl3) (ppm): 7.57-7.55 (1H, m), 7.21-7.17 (3H, m), 5.36 (1H, br), 4.53
(1H, br), 3.80 (3H, s), 3.57 (2H, br), 1.45 (9H, s), 0.92 (9H, s), 0.58 (6H, s).
N-Boc-L-[2-14C]tryptophan methyl ester (10)
A solution of tetra-n-butylammonium fluoride (1 M in THF, 2 mL, 2 mmol) was added
to the solution of 9 (2.04 mmol, 0.89 g, 40.9 MBq) in THF (1.7 mL) and the mixture was
stirred for 10 min. The solution was diluted with ether and the organic layer was separated
and washed with water and dried with Na2SO4. Further purification was carried out using
silica gel chromatography and ethyl acetate:n-hexane (30-70%) as eluent to give the titled
compound 10 (1.82 mmol, 0.58 g, 36.4 MBq) in 89% radiochemical yield. 1H NMR (CDCl3)
(ppm): 8.40(1H, s), 7.59-7.55 (1H, m), 7.37-7.32 (1H, m), 7.24-7.09 (2H, m), 6.96 (1H, s),
5.13 (1H, d), 4.72-4.62 (1H, m), 3.67 (3H, s), 3.30 (2H, d), 1.44 (9H, s)
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L-[2-14C]Tryptophan methyl ester (11)
Compound 10 (1.82 mmol, 0.54 g, 36.4 MBq) was dissolved in DCM (1.5 mL) and
TFA was added and stirred for 1.5 h. The mixture was concentrated in vacuo and the residue
was purified by silica gel column chromatography using ethyl acetate:n-hexane (20-80%) as
eluent to give the titled compound 11 (1.44 mmol, 0.32 g, 28.8 MBq) in 79% radiochemical
1
yield. H NMR (CDCl3) (ppm): 10.12 (1H, s), 7.42 (1H, d), 7.38 (d, 1H), 7.16-7.04 (3H,
m), 4.24 (1H, t), 4.09 (3H, s), 3.66 (3H, s), 3.25 (2H, m)
(2-(acetylthio)acetyl)-L-[2-14C]tryptophan methyl ester (12)
The mixture of 11 (1.44 mmol, 0.32 g, 28.8 MBq), 2-(acetylthio)acetic acid (1.6 mmol,
0.21 g), HBTU (1.58 mmol, 0.6 g), DIPEA (0.43 mmol, 0.06 g) and THF (13 mL) was stirred
overnight. The THF was evaporated in vacuo then ethyl acetate (15 mL) was added. The
organic solution was washed with saturated NaHCO3 (20 mL) and evaporated to give the
crude product. Purification of 12 was carried out by silica gel column chromatography using
ethyl acetate:n-hexane (30-70%) as eluent to give (12) (1.29 mmol, 0.43 g, 25.8 MBq) in
90% radiochemical yield. 1H NMR (CDCl3) (ppm): 8.21 (1H, s), 7.51 (1H, d), 7.32 (1H, d),
7.16-7.00 (3H, m), 6.65 (1H, d), 4.89 (1H, dd), 3.67 (3H, s), 3.49 (2H, dd), 3.29 (2H, m), 2.22
(2H, s).
(2-Mercaptoacetyl)-L-[2-14C]tryptophan (13)
Compound 12 (1.29 mmol, 0.43 g, 25.8 MBq) was dissolved in CH3OH (5 mL), then a
solution of NaOH (5 mL 1 M solution) was added and stirred under nitrogen for one hour.
After evaporation the organic layer in vacuo, water (16 mL) was added and the aqueous
solution was washed with ethyl acetate (30 mL). The pH of the aqueous layer was adjusted to
1 using concentrated HCl and extracted with ethyl acetate. The organic layer was washed
with water and dried (Na2SO4). Filtration and evaporation under reduced pressure gave the
titled compound (0.87 mmol, 0.24 g, 17.3 MBq, %67 radiochemical yield). 1H NMR (MeOD)
(ppm): 7.55 (1H, d), 7.31 (1H, d), 7.13-6.92 (3H, m), 4.72 (1H, dd), 3.36 (1H, dd), 3.22
(1H, dd), 3.13 (2H, s), 1.90 (1H, s).
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Conclusion
In this paper, we have presented a convenient synthetic pathway for labeling of
tryptophan with carbon-14 in the 2-position of indole moiety by using 1H-[2-14C]indole 1, as
a key synthetic intermediate.
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Figure 1- Common structure of beta-lactam antibiotics
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Figure 2- Inhibitors of MBLs
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Figure 3-1H-[2-14C]indole
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Scheme 1. a) 1: H2SO4, 2: LiBEt3H, THF, 76.5%. b) Reflux, 74% c) t-BuOK, t-BuOH, 350
C, 27.2% d) n-BuLi, TBDMSCl, THF, -78 C. quantitave e) NBS, THF, -78 C, 81% f) t-
BuLi, THF, -78 C, 8, 68% g) TBAF, THF, 89% h) DCM, TFA, 79% i) Thioester,
N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate,
O-
(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), N,N-
diisopropylethylamine (DIPEA), THF, 90% j) NaOH, 67%
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