Metabolic Fate of the Isocyanide Moiety: Are Isocyanides Pharmacophore Groups Neglected by Medicinal Chemists?

Article abstract of DOI:10.1021/acs.chemrestox.9b00504

Despite the isolation of hundreds of bioactive isocyanides from terrestrial fungi and bacteria as well as marine organisms, the isocyanide functionality has so far received little attention from a medicinal chemistry standpoint. The widespread tenet that isocyanides are chemically and metabolically unstable has restricted bioactivity studies to their antifouling properties and technical applications. In order to confirm or refute this idea, the hepatic metabolism of six model isocyanides was investigated. Aromatic and primary isocyanides turned out to be unstable and metabolically labile, but secondary and tertiary isocyanides resisted metabolization, showing, in some cases, cytochrome P450 inhibitory properties. The potential therefore exists for the secondary and tertiary isocyanides to qualify them as pharmacophore groups, in particular as war-heads for metalloenzyme inhibition because of their potent metal-coordinating properties.

Full text of DOI:10.1021/acs.chemrestox.9b00504

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Article
The metabolic fate of the isocyanide moiety. Are isocyanides
pharmacophore groups neglected by medicinal chemists?
Ubaldina Galli, Gian Cesare Tron, Beatrice Purghè, Giorgio Grosa, and Silvio Aprile
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00504 • Publication Date (Web): 26 Mar 2020
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The metabolic fate of the isocyanide moiety.
Are isocyanides pharmacophore groups neglected by
medicinal chemists?
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Ubaldina Galli,a Gian Cesare Tron,a Beatrice Purghè,a Giorgio Grosa,a and Silvio Aprilea*
a Department of Pharmaceutical Sciences; Università del Piemonte Orientale; Novara, 28100; Italy
*Corresponding author information
Silvio Aprile - Department of Pharmaceutical Sciences; Università del Piemonte Orientale; Novara,
28100; Italy.
Phone: +39 0321 375855
E-mail: silvio.aprile@uniupo.it
Orcid ID: 0000-0003-4804-9543
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Abstract
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Despite the isolation of hundreds of bioactive isocyanides from terrestrial fungi and bacteria as well
as marine organisms, the isocyanide functionality has so far received scanty attention from a
medicinal chemistry standpoint. The widespread tenet that isocyanides are chemically and
metabolically unstable has restricted bioactivity studies to their antifouling properties and technical
applications. In order to confirm or confute this idea, the hepatic metabolism of six model
isocyanides was investigated. Aromatic and primary isocyanides turned out to be unstable and
metabolically labile, but secondary and tertiary isocyanides resisted metabolization, showing, in
some cases, cytochrome P450 inhibitory properties. The potential therefore exists for the secondary
and the tertiary isocyanides to qualify them as pharmacophore groups, in particular as war-heads for
metalloenzyme inhibition because of their potent metal-coordinating properties.
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Introduction
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Isocyanides (also known as isonitriles or carbylamines) represent a uniqueness in the study of
functional groups in organic chemistry.1 Indeed, due to their resonance form they can behave both
as carbenes and nucleophiles and can easily undergo the so-called α-addition. The passage from a
divalent to a tetravalent status is a highly exothermic and favorable process and it lies at the bottom
of the isocyanide mediated multicomponent reactions.2
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Furthermore, the isocyanide group is a coordinating agent for several metals thanks to its ability to
act as strong σ-type donor.3 At this point, two different scenarios are possible. In the first case, the
complexes are usually stable with the isocyanide group inert to chemical reactions. For example,
they are well-known the strong coordination properties of methylisocyanide with the heme group of
hemoglobin,4 the interaction with both ferric and ferrous iron of the cytochrome P450 (CYP) with
alkylisocyanides,5 the irreversible inhibition of xanthine oxidase by means of ethylisocyanide,6 and
the properties of technetium based contrast agents containing isocyanides which reached the market
with the commercial myocardial perfusion agent Cardiolite® [Tc-MIBI]. In the second case, the
interaction with the metal (e.g. silver and copper), increases the acidity of the hydrogen at the α-
position of the isocyano group toward nucleophiles triggering a plethora of novel transformations
which are regularly reviewed.7 Probably, due to the ease of hydrolysis to formamide under slightly
acidic conditions, chemists were distrustful of isocyanides as potential pharmacophore group to be
used in medicinal chemistry. This idea is also supported by the fact that a leading book as “The
practice of medicinal chemistry” third edition edited by Wertmuth in Chapter 7 lists isocyanides as
metabolic labile pharmacophore groups which should not be used in medicinal chemistry.8
Unfortunately, this statement is not supported by literature references and our search on Sci-Finder
database did not result in any systematic study on the metabolic fate of isocyanides. On the other
hand, in nature hundreds of isocyanides have been isolated and many have demonstrated to possess
biological activity. The intricate structure of some of them, spurred synthetic chemists to attempt
their total synthesis, with a minor interest on the effective biological role played by the isocyanide
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group. Some authors speculated that the isocyanides can be used as a masked war-head which can
be oxidized to isocyanates, strong carbamoylating groups of proteins, responsible for the
antifouling, antibiotic, antifungal and antitumor properties of some of these isocyanide containing
natural products. To this point, some nudibranchs do not synthesize isocyanoterpene as protective
compounds by their own but obtain them from specific sponges used as food source. Anyway, the
picture seems more complex and it is nowadays clear that the isocyanide group plays a role as a real
pharmacophore group. Over the last decades, both natural and synthetic isocyanide containing
molecules have emerged from literature as potent biologically active compounds.9 For example,
xantocillins (A) isolated both from soil and marine fungi, apart their well-recognized antibiotic
properties, showed to be thrombopoietin receptor agonists,10 autophagy inducer with potent anti-
proliferative activity on a panel of human solid tumors,11 and prostaglandin synthesis inhibitors.12
Natural product axisonitrile-3 (B) as many others, showed strong antiplasmodial activity due to the
ability of the isocyanide group to complex the iron atom of heme preventing the crystallization of
hematin to hemozoin.13 Hapalindoles such as C were active as blockers on sodium channels,14 while
kalihinol B (D) was a very potent antimicrobial agent against chloroquine-resistant FCR-3
Plasmodium falciparum.15
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Recently, synthetic isocyanides such as E appeared in medical literature as agents against
multidrug-resistant Staphylococcus aureus,16 and inhibitors of M2 channels in influenza A virus
H5N1 such as F,17 while the antifouling agent G was demonstrated to be selective, able to perturb
only two important enzymes in the aquatic organism B. amphitrite namely a NADH-ubiquinone
oxidoreductase, and cytochrome P450 18 (Figure 1).
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NC
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H
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Figure 1. Selected examples of biologically active isocyanides.
All these results might support the idea of using the isocyanide functional group as a unique
pharmacophore group useful for the discovery and development of novel biologically active
compounds. For this reason, with the intent to evaluate the metabolic stability of the isocyanide
group in the bloodstream, and the occurrence of hepatic metabolism, we studied the in vitro
metabolic transformation of six different isocyanide models (primary, secondary, tertiary aliphatic
isocyanides, aryl and benzylic isocyanides 1-6), (Figure 2).
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Figure 2. Models used to study the isocyanides metabolism.
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Experimental procedures
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Isocyanides and formamides were synthesized according to known procedures (Scheme S1,
Supporting Information). The purity of compounds was determined by high performance liquid
chromatography coupled with ultraviolet-visible detector (LC-UV) using the instrumentation and
methods reported in Supporting Information. Purity of all final compounds was ≥ 95%.
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General. Commercially available reagents and solvents were purchased from Sigma-Aldrich
(Merck) or Alfa Aesar and were used without further purification. Tetrahydrofuran (THF) was
distilled immediately before use from Na/benzophenone under a slight positive atmosphere of dry
nitrogen. Dichloromethane was dried by distillation from P2O5 and stored on activated molecular
sieves (4 Å). When needed the reactions were performed in flame or oven-dried glassware under a
1
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positive pressure of dry nitrogen. H-NMR and C-NMR spectra were recorded on a JEOL ECP
300 MHz spectrometer. Chemical shifts are reported in parts per million (ppm). Mass spectra were
acquired with a Thermo Finningan LCQ-deca XP-plus equipped with an ESI source and an ion trap
detector. High resolution mass spectrometry data were acquired with a Thermo Scientific Q-
Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer. Flash column chromatography
was performed on silica gel (Merck Kieselgel 60, 230–400 mesh ASTM). Thin layer
chromatography (TLC) was carried out on 5  20 cm plates with a layer thickness of 0.25 mm
(Merck Silica gel 60 F254). When necessary they were developed with KMnO4 reagent or
Dragendorff reagent.
Compounds 1A–6A (Scheme S1) were commercially available, while 2-isothiocyanato
naphthalene, and 1-isothiocyanato-1,2,3,4-tetrahydro-napthalene were synthesized as described
previously.19
General Procedure A for the synthesis of formamides (1FA-6FA): Formic acid (103.6 mmol,
7.0 equiv) and acetic anhydride (88.9 mmol 6.0 equiv) were allowed to mix for 1 h at 25 °C, after
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which the appropriate amine (14.8 mmol 1.0 equiv) was added portionwise to the mixture at 0 °C.
The ice bath was removed, and the reaction was stirred at 25 °C until the amine was consumed
completely as determined by TLC (1-3 h). The reaction mixture was diluted with water (100 mL),
the aqueous phase was extracted with CH2Cl2 (2100 mL). The combined organic phases were
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washed with water (170 mL), brine (170 mL), dried over Na2SO4, filtered and concentrated to
give the desired product without purification.
N-(naphthalen-2-yl)formamide (1FA). The title compound was prepared from naphthalen-2-amine
(1A): off-white solid, yield 75%, m.p 132-134 °C; IR (KBr) 3055, 2919, 1728, 1529, 1299, 1279,
849, 827 cm-1; 1H-NMR (300 MHz, CDCl3, mixture of rotamers 1:1): δ 8.85 (d, J = 11.0 Hz, 1-H),
8.58 (br s, 1-H), 8.46 (s, 1-H), 8.22 (s, 1-H), 7.85-7.77 (m, 6-H), 7.67 (br s, 1-H), 7.53-7.39 (m, 6-
H), 7.23 (s, 1-H) ppm; 13C-NMR (75 MHz, CDCl3 mixture of rotamers 1:1): δ 162.9, 161.4, 134.4,
134.3, 133.8, 133.7, 131.2, 130.8, 130.1, 129.0, 127.9, 127.8, 127.7, 127.3, 127.2, 126.8, 125.6,
125.4, 119.7, 118.9, 117.2, 115.3 ppm. MS (ESI): m/z 172 [M + H]+.
N-((1,1'-biphenyl)-4-yl)formamide (2FA). The title compound was prepared from 4-aminobiphenyl
1
(2A): yellowish solid, yield 90%; H-NMR (300 MHz, CDCl3, major rotamer): δ 8.71 (br s, 1-H),
8.45 (s, 1-H), 7.65-7.21 (m, 9-H) ppm; 13C-NMR (75 MHz, CDCl3, major rotamer): δ 159.3, 140.1,
137.8, 136.3, 128.9, 127.8, 127.4, 127.0, 120.5 ppm. MS (ESI): m/z 198 [M + H]+.
N-(2-phenylethyl)formamide (3FA). The title compound was prepared from 2-phenylethanamine
1
(3A): pale yellow oil, yield quantitative; H-NMR (300 MHz, CDCl3, major rotamer): δ 8.03 (s, 1-
H), 7.27-7.16 (m, 5-H), 6.38 (br s, 1-H), 3.51-3.42 (m, 2-H), 2.79-2.77 (m, 2-H) ppm; 13C-NMR (75
MHz, CDCl3, major rotamer): δ 161.7, 138.7, 128.8, 128.7, 126.7, 39.4, 35.5 ppm. MS (ESI): m/z
150 [M + H]+.
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N-(1,2,3,4-tetrahydronaphthalen-1-yl)formamide (4FA). The title compound was prepared from
1,2,3,4-tetrahydronaphthalen-1-amine (4A): off-white solid, yield quantitative; 1H-NMR (300 MHz,
CDCl3, major rotamer): δ 7.94 (s, 1-H), 7.12-6.97 (m, 5-H), 5.0 (s, 1-H), 2.67 (s, 2-H), 1.94-1.67 (m
4-H) ppm; 13C-NMR (75 MHz, CDCl3): δ 160.8, 137.1, 135.8, 128.8, 128.2, 126.9, 125.8, 45.8,
29.7, 28.8, 19.7 ppm. MS (ESI): m/z 176 [M + H]+.
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N-(2-phenylpropan-2-yl)formamide (5FA). The title compound was prepared from 2-phenylpropan-
1
2-amine (5A): pale yellow oil, yield quantitative; H-NMR (300 MHz, CDCl3, major rotamer): δ
7.92 (m, 1-H), 7.80 (br s, 1-H), 7.37-7.24 (m. 5-H), 1.60 (s, 6-H) ppm; 13C-NMR (75 MHz, CDCl3,
major rotamer): δ 166.2, 165.1, 146.3, 128.4, 126.9, 125.0, 30.6 ppm. MS (ESI): m/z 164 [M + H]+.
N-((3S,5S,7S)-Adamantan-1-yl)formamide (6FA). The title compound was prepared from
(3S,5S,7S)-adamantan-1-amine (6A): white solid, yield 90%, m.p. 140-141 °C; IR (KBr) 3439,
2900, 2851, 1696, 1134, 1097, 798, 673 cm-1; 1H-NMR (300 MHz, DMSO, major rotamer): δ 7.82
(d, J = 2.2 Hz, 1-H), 7.59 (br s, 1-H), 2.00 (br s, 6-H), 1.91 (d, J = 2.8 Hz, 6-H), 1.75 (d, J = 2.5 Hz,
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3-H) ppm; C-NMR (75 MHz, CDCl3, major rotamer): δ 162.6, 50.6, 43.8, 35.8, 29.1 ppm. MS
(ESI): m/z 180 [M + H]+.
General Procedure B for the synthesis of isocyanides (1-6): Under a nitrogen atmosphere, a
stirred solution of the appropriate formamide (3.06 mmol, 1.0 equiv) in dry CH2Cl2 (10 mL) at 0 °C
was treated with triethylamine (15.3 mmol 5.0 equiv), followed by POCl3 (3.06 mmol 1.0 equiv)
dropwise. The reaction was allowed to warm to 25 °C while stirring. After 20 min, the reaction was
stopped by the addition of water (30 mL), and the organic layer was extracted with CH2Cl2 (230
mL). The combined organic extracts were washed with water (230 mL), dried over Na2SO4,
filtered and concentrated to give the crude product. Purification by flash column chromatography
PE/EtOAc 95:5 gave the desired isocyanide.
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2-Isocyanonaphthalene (1). The title compound was prepared from N-(naphthalen-2-yl)formamide
(1FA): pale yellow solid, yield 59%, m.p. 63-65 °C dec; IR (KBr) 3064, 2125, 1507, 1438, 1207,
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1152, 894, 821, 754 cm-1; H-NMR (300 MHz, CDCl3): δ 7.83-7.74 (m, 4-H), 7.56-7.53 (m, 2-H)
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7.53 (d, J = 8.9 Hz, 1-H) ppm; C-NMR (75 MHz, CDCl3): δ 164.4, 132.7, 132.5, 129.5, 127.8,
127.7, 127.6, 127.4, 125.6, 123.7 (t, J = 12.9 Hz), 123.1 ppm.
4-Isocyano-1,1ˈ-biphenyl (2). The title compound was prepared from N-((1,1'-biphenyl)-4-
1
yl)formamide (2FA): yellowish-green solid, yield 40%, m.p. 77-78 °C; H-NMR (300 MHz,
CDCl3): δ 7.62-7.56 (m, 4-H), 7.47-7.43 (m, 5-H) ppm; 13C-NMR (75 MHz, CDCl3): δ 164.7,
142.5, 139.4, 129.1, 128.3, 128.1, 127.2, 126.9 ppm.
(2-Isocyanoethyl)benzene (3). The title compound was prepared from N-(2-phenylethyl)formamide
(3FA): yellow-orange oil, yield 51%; 1H-NMR (300 MHz, CDCl3): δ 7.40-7.24 (m, 5-H), 3.58 (t, J
= 6.7 Hz, 2-H), 2.97 (t, J = 6.7 Hz, 2-H) ppm; 13C-NMR (75 MHz, CDCl3): δ 156.4 (t, J = 5.2 Hz),
136.6, 128.6, 128.5, 127.0, 42.8 (t, J = 6.3 Hz), 35.4 ppm.
1-Isocyano-1,2,3,4-tetrahydronaphthalene (4). The title compound was prepared from N-(1,2,3,4-
tetrahydronaphthalen-1-yl)formamide (4FA): yellow oil, yield 50%; 1H-NMR (300 MHz, CDCl3): δ
7.47-7.44 (m, 1-H), 7.30-7.23 (m, 2-H), 7.16-7.14 (m, 1-H), 4.83 ( s, 1-H), 2.93-2.72 (m, 2-H),
13
2.16-2.04 (m, 3-H), 1.89-1.79 (m, 1-H) ppm; C-NMR (75 MHz, CDCl3): δ 155.1, 136.2, 131.9,
129.3, 128.3 (2-C), 126.4, 52.4 (d, J = 5.7 Hz), 30.5, 28.4, 19.1 ppm.
(2-Isocyanopropan-2-yl)benzene (5). The title compound was prepared from N-(2-phenylpropan-2-
1
yl)formamide (5FA): pale yellow oil, yield 60%; H-NMR (300 MHz, CDCl3): δ 7.51-7.32 (m, 5-
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H), 1.79 (s, 6-H) ppm; C-NMR (75 MHz, CDCl3): δ 155.3 (t, J = 5.2 Hz), 142.2, 128.7, 127.8,
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124.1, 60.6 (t, J = 5.7 Hz), 31.9 ppm.
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1-Adamantyl isocyanide (6). The title compound was prepared from N-((3S,5S,7S)-adamantan-1-
yl)formamide (6FA): white solid, yield 76 %, m.p. 185-187 °C (the substance tends to sublimate);
1
IR (KBr) 2914, 2858, 2123, 1454, 1354, 1309, 1103, 1076, 890, 813 cm-1; H-NMR (300 MHz,
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CDCl3): δ 2.07 (br s, 3-H), 2.02 (br s, 6-H), 1.65 (br s, 6-H) ppm; C-NMR (75 MHz, CDCl3): δ
151.6, 54.4, 43.7, 35.6, 28.8 ppm.
Synthesis of N-(naphthalen-2-yl)-5,6-dihydro-1,2,4-dithiazin-3-amine (1TNMr)
2-isothiocyanato naphthalene was prepared from naphthalen-2-amine (1A) according to the
literature method (Scheme S2).19
To a stirred solution of 2-mercaptoethylamine hydrochloride (0.061 g, 0.54 mmol, 1 equiv) and
triethylamine (0.075 mL, 0.54 mmol, 1 equiv) in dry THF (1.0 mL), 2-isothiocyanato naphthalene
(0.10 g, 0.54 mmol, 1 equiv) dissolved in dry THF (1.0 mL) was added dropwise, under nitrogen
atmosphere. The reaction mixture was stirred overnight at room temperature, then it was
concentrated under reduced pressure and the crude material was purified by column
chromatography using PE/EtOAc 8:2, EtOAc and EtOAc/MeOH 9:1 as eluents to give the disulfide
intermediate as an off-white solid, yield 76%, 1H-NMR (300 MHz, DMSO): δ 9.8 (br s, 1-H), 8.04
(br s, 1-H), 7.93-7.82 (m, 4-H), 7.51-7.44 (m, 3-H), 3.83 (br d, 2-H), 3.01 (t, J = 6.2 Hz, 2-H) ppm;
13C-NMR (75 MHz, DMSO): δ 180.6, 136.6, 133.3, 130.4, 128.3, 127.5, 127.4, 126.3, 125.2, 123.6,
119.8, 43.1, 36.5 ppm.
A solution of disulfide intermediate (0.043 g, 0.081 mmol, 1 equiv), DL-dithiothreitol (DTT)
(0.0375 g, 0.243 mmol, 3 equiv) and two drops of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as
catalyst in 3.0 mL CH2Cl2 was stirred at reflux for 2.5 h. Then, the solution was washed with water
(2), brine (1), dried over Na2SO4 and concentrated in vacuo to afford product as a pale yellow
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solid, yield 54%, H-NMR (300 MHz, CDCl3): δ 8.34, (br s, 1-H), 7.92-7.72 (m, 4-H), 7.54-7.47
5
6
(m, 2-H), 7.33 (d, J = 8.5 Hz, 1-H), 6.66 (br s, 1-H), 3.82 (q, J = 5.8 Hz, 2-H), 2.80 (q, J = 5.8 Hz,
7
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13
2-H) ppm; C-NMR (75 MHz, CDCl3): δ 180.8, 133.9, 133.4, 132.1, 130.6, 128.0, 127.9, 127.4,
9
126.8, 123.5, 123.2, 47.9, 24.3 ppm. MS (ESI): m/z 180 [M + H]+. MS (ESI): m/z 263 [M + H]+.
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Synthesis of 2-aminoethyl-(1,2,3,4-tetrahydronaphthalen-1-yl)carbamodithioate (4TSM)
1-Isothiocyanato-1,2,3,4-tetrahydro-napthalene was prepared from 1,2,3,4-tetrahydronaphthalen-1-
amine (4A) according to the literature method (Scheme S3).19
To a stirred solution of 2-mercaptoethylamine hydrochloride (0.060 g, 0.53 mmol, 1 equiv) in
CH2Cl2 (2.0 mL), 1-isothiocyanato-1,2,3,4-tetrahydro-napthalene (0.10 g, 0.53 mmol, 1 equiv)
dissolved in CH2Cl2 (1.0 mL) was added dropwise, under nitrogen atmosphere, followed by 5 drops
of N,N-dimethylformamide The reaction mixture was stirred overnight at room temperature, then it
was concentrated under reduced pressure and the crude material was purified by column
chromatography using EtOAc/MeOH 9:1 as eluent to give the desired product as an off-white solid,
yield 67%, 1H-NMR (300 MHz, CDCl3): δ 8.48 (br s, 1-H), 7.26-7.07 (m, 4-H), 5.82, (s, 1-H), 3.65
(br s, 2-H), 3.26 (br s, 2-H), 2.86-2.68 (m, 2-H), 2.04-1.81 (m, 4-H) ppm; 13C-NMR (75 MHz,
CDCl3): δ 194.5, 137.8, 135.1, 129.4, 128.9, 127.7, 126.4, 55.7, 39.9, 32.5, 29.2, 28.7, 20.5 ppm.
MS (ESI): m/z 267 [M + H]+.
LC-UV analyses
A Shimadzu HPLC system (Shimadzu, Kyoto, Japan), consisting of two LC-10AD Vp module
pumps, a SLC-10A Vp system controller, a SIL-10AD Vp autosampler and a DGU-14-A on-line
degasser, was used for the analysis. All the chromatographic separations were performed on a
Kinetex C18 XB 150  4.6 mm (5 μm d.p.) column as a stationary phase protected by a C18-
Security Guard™ (Phenomenex, Torrance, CA). The SPD-M10Avp photodiode array detector was
used to detect the analytes at 254 and 280 nm. LC Solution 1.24 software was used to process the
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chromatograms. Aliquots (20 μL) of the supernatants obtained from the incubations were injected
onto the HPLC system. The mobile phase (flow rate 1 mL/min) consisted of solvent A 0.5% formic
acid aqueous solution and solvent B acetonitrile. The following gradient elution programs were
used: a) 0 min B=20%, 12.5 min B=70%, 14.5 min B=70%, 15 min B=20%, 20 min B=20% - for
incubations of compounds 1, 3-5; b) 0 min B=20%, 12.0 min B=80%, 14.5 min B=80%, 15 min
B=20%, 20 min B=20% - for incubations of compound 2. The eluents were filtered through a 0.45-
µm pore size nylon membrane filter before use.
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LC-MSn analyses
A Thermo Finningan LCQ Deca XP plus system equipped with a quaternary pump, a Surveyor AS
autosampler, a Surveyor photodiode array detector and a vacuum degasser was used for LC-MS
analyses (Thermo Electron Corporation, Waltham, MA). All the chromatographic separations were
performed on a Sinergy Polar 150  2 mm (4 μm d.p.) column protected by a Polar RP-Security
Guard™ (Phenomenex, Torrance, CA) kept at a 35 °C. Aliquots (5 μL) of supernatants obtained
from incubations were injected onto the system and eluted with a mobile phase (flow rate 250
µL/min) consisting of solvent A 0.2% formic acid aqueous solution and solvent B 0.2% formic acid
in methanol. The following gradient elution programs were used: 0 min B=20%, 6 min B=70%, 10
min B=80%, 10.5 min B=20%, 15.5 min B=20% for 1 and 2; 0 min B=15%, 7 min B=70%, 10 min
B=80%, 10.5 min B=15%, 15.5 min B=15% for 3 and 4.
The eluents were filtered through a 0.45-µm pore size nylon membrane filter before use. The eluate
was injected into the electrospray ion source (ESI) and MS spectra were acquired and processed
using Xcalibur® software. The operating conditions of the ion trap mass spectrometer were as
follows: positive mode, spray voltage, 5.20 kV; source current, 80 μA; capillary temperature, 300
°C; capillary voltage, 7.00 V; tube lens offset, 5.00 V; multipole 1 offset, -5.00 V; multipole 2
offset, -11.50 V; sheath gas flow (N2), 40 Auxiliary Units; Auxiliary sheath gas flow, 4 Auxiliary
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Units. Data were acquired in full-scan and product ion scan modes (MSn) using mass scan range m/z
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105-800. The collision energy was optimized at 28-32%.
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Rat and human liver preparations
Male Wistar Han rat liver microsomes (RLM), (protein concentration: 20 mg/mL, total CYP: 650
pmol/mg protein), human liver microsomes (HLM), (pooled mixed sex, fifty individual donors,
protein concentration: 20 mg/mL, total CYP: 360 pmol/mg protein) were purchased from Corning
B.V. Life Sciences (Amsterdam, The Netherlands). All the incubations were performed using a
horizontal DUBNOFF shaking thermostatic bath (Dese Lab Research, Padova, Italy) while
protecting from light.
Incubations with RLM, HLM, recombinant CYP3A4
The standard incubation mixture (250 µL final volume), was carried out in a 50 mM
tris[hydroxymethyl]aminomethane (TRIS)/HCl buffer (pH 7.4) containing 3.3 mM MgCl2, 1.3 mM
NADPNa2, 3.3 mM glucose 6-phosphate, 0.4 Units/mL glucose 6-phosphate dehydrogenase,
acetonitrile as cosolvent (1% of total volume), and the substrate (100 μM). After pre-equilibration
of the mixture, an appropriate volume of RLM or HLM suspension was added to give a final protein
concentration of 1.0 mg/mL. The mixture was shaken for 60 minutes at 37 °C. Control incubations
were carried out without the presence of NADPH-regenerating system or microsomes. Each
incubation was stopped by the addition of 250 µL ice-cold acetonitrile, vortexed and centrifuged at
13000 rpm for 10 min. The supernatants were analyzed by LC-UV or LC-MS. Alternatively, when
the chemical reactivity of the metabolites was studied, the following trapping agents (at 3 mM
concentration) were added to the incubation mixture: glutathione in its reduced (GSH) or oxidised
(GSSG) forms, N-acetylcysteine (NAC), and mercaptoethylamine (MEA).
Incubations with human hemoglobin
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The standard incubation mixture (500 µL final volume), was carried out in a 50 mM
tris[hydroxymethyl]aminomethane (TRIS)/HCl buffer (pH 7.4) containing acetonitrile as cosolvent
(1% of total volume), human hemoglobin (lyophilized powder from Sigma Aldrich, final protein
concentration 1 mg/mL), GSH (3 mM), and the substrate (100 μM). The mixture was shaken for 60
minutes at 37 °C. Control incubations were carried out without the presence of hemoglobin or GSH.
Each incubation was stopped by the addition of 500 µL ice-cold acetonitrile, vortexed and
centrifuged at 13000 rpm for 10 min.
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Incubations with rhodanese
The standard incubation mixture (250 µL final volume), was carried out in a 50 mM borate buffer
(pH 8.6) containing 0.25 mM sodium thiosulfate, acetonitrile as cosolvent (1% of total volume), and
the substrate (200 μM). After pre-equilibration of the mixture, an appropriate volume of rhodanese
from bovine liver (Type II, lyophilized powder from Sigma Aldrich) was added to give a final
protein concentration of 1.0 mg/mL. The mixture was shaken for 60 minutes at 37°C. Control
incubations were carried out without the presence of sodium thiosulfate or rhodanese or trapping
agent (GSH or MEA). Each incubation was stopped by the addition of 250 µL ice-cold acetonitrile,
vortexed and centrifuged at 13000 rpm for 10 min. The supernatants were analyzed by LC-UV or
LC-MS.
CYP inhibition: aminopyrine N-demethylase assay
Aminopyrine N-demethylase activity was determined by detecting the amount of formaldehyde
produced by RLM. The incubation was carried out in Tris/HCl buffer (50 mM, pH 7.4)
supplemented with 150 mM KCI and 10 mM MgCl2. The incubation contained 1 mM aminopyrine,
acetonitrile (1% final volume), and 1.0 mg/mL of RLM in a total volume of 180 µL. Increasing
concentrations (0.01, 1, 10, 25, 50, 75, 100 µM) of the tested isocyanides or known CYP inhibitors
(ketoconazole and quinidine) were added to the incubation mixture. After pre-incubation of the
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assay media for 3 min at 37 °C, the reaction was initiated by adding NADPH (final concentration
1mM) and carried out at 37 °C for 15 min with moderate shaking. The reaction was then terminated
by addition of 90 µL of 20% (w/v) TCA solution. After centrifugation at 13000 rpm for 10 min, a
240 µL aliquot of the protein-free supernatant was treated with 120 µL of Nash reagent and
incubated in a water bath at 37 °C for 40 min. The absorbance of the resultant solution was
determined at 412 nm, subtracting the blank sample absorbance (without NADPH); the
concentration of formaldehyde was quantified (nmol/min/mg) by comparison with a standard curve
prepared from commercially available formaldehyde solution freshly standardized by iodometric
titration. All incubations were performed in triplicate.
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Nash reagent:20 3.75 g ammonium acetate, 50 mL glacial acetic acid, and 75 mL acetylacetone were
dissolved in 25 mL deionized water. The solution was freshly prepared and used for one day.
Determination of the isocyanides reactivity toward protein sulphydryl groups (Ellman’s assay)
To a 2140 µL of 100 mM phosphate buffer (pH 8.0) were added 60 µL of methanol and 750 µL of
bovine serum albumin (BSA) stock solution (lyophilized powder from Sigma Aldrich, 10.1 mg/mL
- 151.2 µM, final concentration 37.8 µM). Ellman’s reagent (50 µL), (15 mM DTNB dissolved in
50 mM ammonium acetate) was added and the solution was incubated at room temperature for 5
minutes before measuring the development of absorbance at 412 nm. When reactivity of 1 and 2
toward BSA sulphydryl groups was assessed, 60 µL of the methanolic solutions of the isocyanides
(final concentration 200 µM) were added in the mixture 10 min before adding the Ellman’s reagent.
Positive control assays were performed by adding NEM (final concentration: 200 µM) instead of
the isocyanides.
The thiol molar concentration [SH] was calculated according to the Eq. 1 where Ai is the
absorbance of the isocyanides incubations, Ab is the absorbance of isocyanides (measured in the
absence of DTNB), and 14150 M-1 cm-1 is the extinction coefficient of the reagent.21
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퐴푖 ―퐴푏
[SH] =
14150 1cm

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Results
Isocyanides incubations in liver microsomes.
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The hepatic metabolism of the selected isocyanides was investigated through in vitro incubations in
rat (RLM) and human (HLM) liver microsomes supplied by a NADPH-regenerating system.
Reactivity toward nucleophiles was then investigated by supplying the following trapping agents:
reduced (GSH) and oxidised (GSSG) glutathione, N-acetylcysteine (NAC), and
mercaptoethylamine (MEA).
The incubations of 1 and 2 were analysed by both LC-UV and LC-MSn systems; since their poor (3-
5) or absent (6) UV-Vis absorption property, these isocyanides were investigated by LC-MSn only.
Microsomal metabolism of 2-isocyanonaphthalene (1) and 4-isocyano-1,1'-biphenyl (2).
Incubations of aryl isocyanides 1 and 2, performed in RLM supplied with GSH without NADPH,
gave the formation of three main metabolites (1A, 1FA, 1NG, and 2A, 2FA, 2NG respectively)
observed in LC-UV chromatograms with the substrates disappearing up to 75% after one hour
(Figure 3a). When the aryl isocyanides were incubated in RLM supplied with GSH and in the
presence of NADPH, an additional metabolite (1SG or 2SG) was detected (Figure 3b). Control
incubations of 1 and 2, in the presence of GSH only, did not give any transformation.
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Figure 3. LC-UV chromatograms of isocyanides 1 and 2 incubations in RLM supplied by GSH:
without NADPH (a), with NADPH (b).
The structures of metabolites from 1 and 2 were investigated by LC-tandem mass spectrometry. The
samples were analysed in positive full scan and product ion scan (MSn) modes revealing the
formation of the arylamines 1A (m/z 144), 2A (m/z 170), and the formamides 1FA (m/z 172), 2FA
(m/z 198) respectively; their structures were confirmed by chromatographic comparison with the
synthetized reference compounds (Figure 4). In addition, formation of the formamidine adducts
1NG (m/z 461) and 2NG (m/z 483) deriving from α-addition between the amino group of GSH on
isocyanide were observed (Figure 4). For both the adducts, 1NG and 2NG, the protonated
molecules at m/z 461 and 487 corresponding to the addition of 307 Da, alongside the MSn data
suggested the equimolar reaction between the isocyanide and the amino group of GSH. Indeed, their
MS2 product ion spectra showed a main fragment at m/z 283 or 309 respectively, corresponding to
the neutral loss of the Cys-Gly dipeptide (178 Da). Further fragmentation (MS3) of these ions
formed the main fragments at m/z 154 and 130 and 180 and 130 respectively which corroborated the
proposed structures of metabolites (Figure 4), (See figures S2-S5 for spectral data).
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N
NH2
O
H
N
H
O
NH2
8
9
2A
(m/z= 170)
2FA
(m/z= 198)
1FA
(m/z= 172)
1A
(m/z= 144)
144
170
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+
+
MS3
H
HOOC
MS3
H
N
HOOC
283
N
O
309
O
130
130
154 (MS3 283)
180 (MS3 309)
N
SH
N
SH
O
H
O
H
HN
N
COOH
HN
N
COOH
N
H
N
COOH
O
H
COOH
O
2NG
(m/z= 487)
1NG
(m/z= 461)
HN
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H
HN
O
S
S
O
O
H
N
H2N
N
COOH
H2N
COOH
N
N
H
H
COOH
O
COOH
O
2SG
(m/z= 503)
1SG
(m/z= 477)
374
348
MS3
MS3
MS3
MS3
+
+
SH
SH
-H2O
-H2O
H
H
356
330
N
COOH
N
COOH
H2N
H2N
O
O
179
179
-NH3
162
-NH3
162
144
144
-H2O
-H2O
Figure 4. Structures and MSn fragmentation pathways of metabolites from the isocyanides 1 and 2.
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For the additional metabolites 1SG (m/z 477) and 2SG (m/z 503), which were formed under
microsomal oxidative conditions, the mass shift of 16+307 Da suggested the formation of a
thiocarbamate analogue deriving from the reaction between the thiol group of GSH and the
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isocyanate intermediate formed by oxidation of isocyanide (Figure 4). The MS2 spectra of these
ions showed the main fragments at m/z 348 and 374 respectively (neutral loss of pyroglutamic acid,
129 Da) which further fragmentation (MS3) generated four ions at m/z 330, 179, 162, and 144 for
1SG, and 356, 179, 162, and 144 for 2SG (Figure 4 and Figures S2-S5). It is worth of mention that
similar adducts, associated with the same fragmentation pathways, have been previously reported
for phenyl isocyanate (-N=C=O) adducts with GSH.22 For confirmatory purpose, the molecular
masses of 1NG, 1SG, 2NG, 2SG adducts were also confirmed by high resolution mass
spectrometry (See Figures S6-S7 for experimental data).
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Furthermore, the reactivity of isocyanide 1 was studied in RLM activated by NADPH and in the
presence of other trapping agents such as: GSSG, NAC, and MEA. Incubation supplied by GSSG
gave rise to the metabolite 1NGG (m/z 766) and, similarly to incubation with GSH, both 1FA and
1NA were also formed. Reactions with NAC or MEA, only generated 1SN (m/z 333) and 1SM (m/z
247) via cysteinyl thiol nucleophilic attack, whilst both 1FA and 1NA were absent (Scheme 1) (See
Figures S8-S9 for structures and spectral data). The structures of these three adducts were
confirmed by the presence of several diagnostic ions detected in their positive product ion spectra.
1NGG gave two abundant ions: m/z 637 (neutral loss of pyroglutamic acid, 129 Da) and m/z 623
(formamidine cleavage) which were submitted to further fragmentation stage (MS3). The diagnostic
MS3 fragment ions, m/z 548 (loss of glycine), 494 (formamidine cleavage and loss of pyroglutamic
acid), and 355 (amide cleavage) confirmed the proposed structures for these metabolites. Finally,
product ion scan spectrum of isocyanide 1 adduct with NAC, 1SN (m/z 333) gave an abundant ion
at m/z 164 (acetylcysteine loss) which gave, when submitted to MS3 stage, two abundant ions at m/z
146 and 122 corresponding to neutral losses of water and the acetyl group. Similarly, isocyanide 1
reacted with MEA to form the metabolite 1SM (m/z 247) which fragmentation pathways gave rise
to the diagnostic ions at m/z 230 (loss of ammonia), 170 (mercaptoethylamine cleavage), and 144
(N-C cleavage) (See Figure S9 for spectral data and structures).
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In order to demonstrate the metabolic pathway leading to the formation of amine 1A, an additional
incubation of formamide 1FA was carried out in the presence of RLM activated by NADPH and
supplied with GSH. The same peaks attributable to 1A and 1SG, revealed in isocyanide 1
incubations, were detected (Figure S10). This means that N-aryl formamide 1FA could be oxidized
to isocyanate which can either react with GSH to give 1SG or with water to give 1A after
spontaneous decarboxylation of carbamic acid. Alternatively, it could be deacylated in liver
microsomes giving the corresponding amine 1A (Scheme 1).
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Finally, adduct 1NG was isolated by semipreparative liquid chromatography: on standing, it
spontaneously hydrolysed to 1FA. (Figure S11).
Differently from incubations in rat and human liver microsomes, incubation of 1 in the presence of
recombinant CYP3A4 isoform supplied by NADPH and GSH only gave small traces of 1NG and
1SG adducts, whereas formamide 1FA and amine 1A were absent (data not shown): however, these
data were obtained from a single CYP isoform, therefore, if on the one hand, it is not possible to
exclude the role of other CYPs in the formation of isocyanide adducts; on the other hand, further
metalloenzymes or enzymes present in the microsomal fraction could actively participate in the
biotransformation of isocyanides.
Overall, the metabolic pathways summarized in Scheme 1 demonstrate that aryl isocyanides 1 and 2
are not stable in in vitro metabolism; indeed, in liver they are prone to react with nucleophilic
agents: mainly via direct nucleophilic attack of the glutamic acid α-amino group of GSH and, in a
lesser extent, via the cysteinyl thiol of GSH after oxidation to isocyanate intermediate.
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NC
NC
H
N
S
R-SH
microsomes + NADPH
R
1
2
Ar
Ar-N=C=O
O
1SG
2SG
1SN
1SM
(m/z 333), (m/z 247),
(m/z 477),
(m/z 503)
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microsomes
+ NADPH
microsomes + R-NH2
microsomes+ NADPH
H
N
NH2
O
N
Ar
Ar
Ar
HN
R
1A
(m/z 144)
1FA
2FA
(m/z 172)
(m/z 198)
1NG
2NG
1NGG
(m/z 461),
(m/z 487)
(m/z 766),
2A
(m/z 170)
Scheme 1. Proposed in vitro metabolic pathways of aryl isocyanides 1 and 2. (Dashed lines indicate
non enzymatic transformations; structure in parenthesis indicates undetected intermediate; R
indicates: reduced and oxidised glutathione, N-acetylcysteine, and mercaptoethylamine scaffolds).
Microsomal metabolism of (2-isocyanoethyl)benzene (3).
Incubations of 3 was performed in RLM activated by NADPH and supplied with nucleophilic
trapping agents. When incubated with GSH, 3 formed in traces the adduct 3NG (m/z 439) and
formamide 3FA (m/z 150), thus suggesting a limited substrate depletion; furthermore, the adduct
3SG (m/z 455) was also revealed (Scheme 2 and Figure S12). Even, when 3 was incubated in the
absence of NADPH, only traces of 3NG were revealed (data not shown). Hence, for isocyanide 3,
reaction with GSH occurred either via nucleophilic attack by the cysteinyl thiol or the α-amino
group of glutamic acid in a similar manner but to a minor extent with respect to aryl isocyanides 1
and 2. The structures of these two adducts were confirmed by the presence of several diagnostic
ions revealed in their MSn spectra (Figure S12). Indeed, MS2 spectrum of 3NG showed an abundant
fragment ion at m/z 261 (neutral loss of Cys-Gly dipeptide, 178 Da) which further fragmentation
(MS3) gave two prominent fragment ions at m/z 130 (loss of pyroglutamic acid), and 105 (N-C
cleavage). 3SG metabolite also showed an abundant fragment ion at m/z 326 (loss of pyroglutamic
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acid) which, when submitted to MS3 stage, gave four fragment ions being the most abundant at m/z
179 due to the loss of the Cys-Gly dipeptide.
6
7
8
9
Microsomal incubation of 3 activated by NADPH in the presence of NAC or MEA generated both
the adducts 3SN (m/z 311) and 3SM (m/z 225) exhibiting only traces of formamide 3FA (m/z 150),
(Scheme 2 and Figure S13). Product ion scan spectrum of 3SN gave the same intense ion at m/z 164
(acetylcysteine loss) already observed for 1SN: MS3 stage gave the same two abundant ions at m/z
146 and 122 corresponding to neutral loss of water and the acetyl group respectively. 3SM gave rise
to three abundant and diagnostic ions at m/z 208 (loss of ammonia), 148 (loss of
mercaptoethylamine) and 78 (protonated mercaptoethylamine). No adducts were detected when 3
was incubated in the presence of GSSG trapping agent.
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These data (summarized in Scheme 2) indicate that isocyanide 3 shares the same metabolic
pathways of aryl isocyanides 1 and 2.
O
N
H
N
NC
C
S R
R-SH
microsomes + NADPH
O
3SG
3SN
3SM
(m/z 311), (m/z 225)
(m/z 455),
3
microsomes + R-NH2
N
NH
R
N
O
H
3NG
(m/z 439)
3FA
(m/z 150)
Scheme 2. Proposed in vitro metabolic pathways of isocyanide 3. (Dashed line indicates non
enzymatic transformation; structure in parenthesis indicates undetected intermediate; R indicates:
reduced glutathione, N-acetylcysteine, and mercaptoethylamine scaffolds).
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4
5
6
Microsomal metabolism of 1-isocyano-1,2,3,4-tetrahydronaphthalene (4), (2-isocyanopropan-2-
yl)benzene (5), and adamantyl isocyanide (6).
7
8
9
When compounds 4 and 5 were incubated in RLM activated by NADPH and supplied with GSH or
MEA, the adducts 4SG (m/z 481) and 5SG (m/z 469) or 4SM (m/z 251) and 5SM (m/z 239) were
formed respectively, (Scheme 3), (see Figure S14 for structures and spectral data). This
demonstrates that secondary and tertiary aliphatic isocyanides only react with the nucleophilic thiols
via isocyanate intermediate, being absent the adducts with the amino groups of GSSG and NAC and
also the formamides and amines revealed for the other isocyanides. This strongly suggests their
high hepatic metabolic stability. Adamantyl isocyanide 6 revealed to be even more metabolically
stable, only giving traces of the adduct 6SG (m/z 485), (Scheme 3 and Figure S15).
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NC
H
4
microsomes + NADPH
R-SH
N
S
R
N
C O
NC
NC
O
4SG
5SG
5SM
6SG
(m/z 485),
(m/z 481),
(m/z 251),
(m/z 469),
R-NH2
4SM
(m/z 239)
6
5
N
H
NH2
O
N
HN
R
Scheme 3. Proposed in vitro metabolic fate of isocyanides 4, 5, 6. (Structure in parenthesis indicates
undetected intermediate; R indicates: reduced glutathione and mercaptoethylamine scaffolds).
Isocyanides incubations with rat plasma and human hemoglobin.
After identifying the metabolic pathways involved in the isocyanide hepatic degradation, we
investigated their blood stability, a mandatory condition to consider isocyanides as potential drug
candidates. This was done through an indirect way, evaluating the stability of all the isocyanides
both in plasma and in human hemoglobin.
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They were incubated at 1 mM concentration for 1 hour in plasma or human hemoglobin and the
residual substrate evaluated by LC-UV analysis. Overall, all the isocyanides resulted stable in these
conditions except 1 and 2; when incubated with human hemoglobin supplied with GSH, they gave
the same adducts detected in rat and human liver microsomes: 1-2SG and 1-2NG (the latter present
in traces), (Scheme 1), (data not shown). These results suggested that hemoglobin can
spontaneously catalyse the same reactions catalyzed in liver microsomes: the direct nucleophilic
attack of the glutamic acid α-amino group, and the cysteinyl thiol of GSH via isocyanate
intermediate.
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60
Isocyanides incubations with bovine liver rhodanese.
As rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) is a hepatic enzyme involved in the direct
detoxification of cyanide ions forming thiocyanates, we were intrigued to whether isocyanides
might react with this enzyme.
Experiments with 2-isocyanonaphthalene (1) and 4-isocyano-1,1'-biphenyl (2).
Incubation of isocyanides 1 or 2 with rhodanese in the presence of sodium thiosulfate and supplied
with GSH gave rise to the formation of the thiourea adducts 1TSG (m/z 493) or 2TSG (m/z 519)
respectively, suggesting the formation of a GSH adduct via isothiocyanate intermediate (scheme 4).
The MS2 spectrum of 1TSG showed a main fragment at m/z 364 (neutral loss of pyroglutamic acid,
129 Da) and other diagnostic ions at m/z 418, 330, 289, 22 and 144 which structures were proposed
in figure S16. Similar fragmentations occurred for 2TSG (Figure S18). Alternatively, incubation of
1 in the presence of MEA gave the adduct 1TNM (m/z 261) as a result of the nucleophilic attack via
isothiocyanate intermediate by the amino group of MEA. 1TNM, which is featured by the
fragmentation pathways reported in Figure S17 was supposed to be generated from thiourea
intermediate oxidation to give the corresponding disulfide derivative. To support this hypothesis,
incubation of 1 was treated with dithiothreitol (DTT); as the results 1TNM peak intensity decreased
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and a new peak 1TNMr (m/z 263) was observed. Product ion scan spectrum of m/z 263 gave the
fragmentation pathways depicted in Figure S17 supporting the structure of the metabolite which
was definitively confirmed by comparison of its retention time and spectral data with those of a
synthetic reference compound.
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NC
Ar
HN
S
1
rhodanese
GSH
S
NC
O
Ar
N
C S
H
N
2-
S2O3
H2N
COO
N
H
H
COOH
O
MEA
2
1TSG
2TSG
(m/z 493),
(m/z 519)
Ar
HN
Ar
S
NH
S
N
S
HN
1TNM
(m/z 261)
SH
1TNMr
(m/z 263)
Scheme 4. In vitro metabolic pathways of isocyanides 1 and 2 in the presence of rhodanese.
(Dashed lines indicate non enzymatic transformation; structures in parenthesis indicate undetected
intermediate).
Experiment with (2-isocyanoethyl)benzene (3) and 1-isocyano-1,2,3,4-tetrahydronaphthalene (4).
Incubation of 3 with rhodanese in the presence of GSH gave rise to metabolic transformations
similar to those of compound 1 albeit to a lesser extent. (Scheme 5). Rhodanese catalysed the
formation of isothiocyanate intermediate of 3, which reacted with GSH or MEA via thiol or amino
nucleophilic attack respectively to form the adducts 3TSG (m/z 471) and 3TNM (m/z 239) (Scheme
5); these structures and fragmentation pathways are depicted in Figure S19. Also, for 4, rhodanese
catalysed the formation of 4TSG, (m/z 497) and 4TSM, (m/z 267) (Scheme 5). The presence of the
diagnostic fragment ion at m/z 368 (neutral loss of pyroglutamic acid, 129 Da) in 4TSG MS2
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spectrum confirmed that the nucleophilic attack of GSH via SH group occurred. Differently,
nucleophilic attack of MEA on 4 isothiocyanate occurred via SH group instead of NH2 to form
4TSM; its structure was unequivocally confirmed by the diagnostic ion fragments and identical
retention time with respect to the synthetic reference compound (Figure S20).
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NC
HN
S
rhodanese
GSH
N C S
S
O
2-
S2O3
H
N
H2N
COO
H
N
3
H
COOH
O
MEA
3TSG
(m/z 471)
HN
HN
S
NH
S
S
N
SH
3TNM
(m/z 239)
3TNMr
(m/z 241)
NC
HN
S
rhodanese
GSH
N C S
S
2-
O
S2O3
H
H2N
N
COO
H
N
4
H
COOH
O
MEA
4TSG
(m/z 497)
HN
S
HS
NH2
4TSM
(m/z 267)
Scheme 5. In vitro metabolic pathways of isocyanides 3, 4 with rhodanese. (Dashed lines indicate
non enzymatic transformation; structure in parenthesis indicates undetected intermediate).
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Cytochrome P450 inhibition: aminopyrine N-demethylase assay
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8
9
To evaluate the CYPs inhibition by means of isocyanides 1-6, the aminopyrine N-demethylase
assay was performed.23 The results indicated that both aromatic and the aliphatic isocyanides can
inhibit CYPs with an IC50 comprised in a micromolar range (0.5 – 32.1 µM), (Table 1). However,
except for 3 and 6, the isocyanides inhibited CYPs more efficiently with respect to the inhibitors
used as a control (i.e. ketoconazole for CYP3A, and quinidine for CYP2D6) which in our assay
gave IC50 = 14.0 and 12.1 µM respectively.
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Isocyanide 1 reactivity toward BSA sulphydryl groups
Taking into account the above described reactivity of the isocyanide moiety toward nucleophilic
functions during the metabolism, we evaluated its ability to react with the free cysteine SH residues
of bovine serum albumin (BSA) using the previously reported24 spectrophotometric assay based on
Ellman’s reagent. The results expressed as residual [SH]/[BSA] molar ratio demonstrated the
inability of isocyanides 1 and 2 to bind the free SH group of BSA in comparison to the positive
control N-ethylmaleimide (NEM) which reduced the ratio from 0.215 to 0.021 (Table 1).
CYP inhibition
Ellman’s assay
compound
IC50 µMa,b
3.2
[SH]/[BSA]c
1
2
3
4
5
6
0.172
0.230
2.1
32.1
3.6
0.5
19.3
-
-
-
-
Table 1.
a
IC50 for aminopyrine N-demethylase inhibition was determined from the formaldehyde formation velocity
(nmol/min/protein mg) versus log isocyanides concentration (0.01-100 µM) using GraphPad PRISM 6.0 software.
b
Control incubations with known CYP inhibitors, gave IC50 values of 14.0 µM for ketoconazole (CYP3A) and 12.1
µM for quinidine (CYP2D6).
c Control incubation without isocyanides [SH]/[BSA]= 0.215; positive control with NEM [SH]/[BSA]= 0.021
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Discussion
6
7
8
9
In this work, we investigated the metabolic stability of the isocyanide functional group by studying
the metabolic profile of some aryl (1-2) and aliphatic (3-6) isocyanides models.
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Depending on their structural features, the metabolic stability of drugs could be strictly related to
their chemical stability; hence, the integration of both aspects could be conveniently performed
defining a unique chemical and metabolic stability space. For this reason, chemical stability of
isocyanides in aqueous media at physiological conditions was first investigated confirming
substantially no degradation. Similar results were obtained in preliminary plasma and hemoglobin
solution stability studies. Next, exploiting the sensitivity and the power of electrospray ionization
tandem mass spectrometry (ESI-MSn) for structural characterization, we elucidated the structures of
isocyanides metabolites generated in liver microsomes supplied with nucleophilic trapping agents
(e.g. GSH). Indeed, we first supposed that the interaction with metal containing enzymes (e.g.
CYPs), may increases the reactivity of the isocyanide group toward nucleophiles triggering a
plethora of biotransformations.
Our results proved that both aryl and aliphatic isocyanides undergo hepatic metabolism. However,
if the aryl isocyanides 1 and 2 and the primary aliphatic isocyanide 3, reacting with nucleophilic
trapping agents, undergo an extensive biotransformation in liver microsomes, (Scheme 1-2),
secondary and tertiary isocyanides are stable, undergoing only a mild NADPH dependent
microsomal oxidation. Both aryl isocyanides 1-2 and the primary aliphatic isocyanide 3 react via
the direct nucleophilic attack of the -NH2 of GSH to give the formamidine adducts, which can be
partially hydrolysed into formamide.
A possible mechanism to account for the formation of 1NG has to consider the involvement of a
metal catalyst. Indeed, it is well-know that, even at temperature higher than 100 °C neither amines
nor thiols can give α-addition with the isocyano moiety.1b This reaction usually requires the
presence of a transition metal of group IB or IIB. In particular, isocyanides coordinate the metal to
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