Phenylsulfonylnitromethanes as potent irreversible inhibitors of aldose reductase

Article abstract of DOI:10.1016/S0223-5234(99)00219-6

Aldose reductase (AR) inhibition provides a viable pharmacologically direct mode for the treatment of diabetic complications. We have synthesized a series of N-4 substituted analogues (15-21) of the known aldose reductase inhibitor phenyl-sulfonylnitromethane. The compounds are potent inhibitors of AR with IC₅₀s between 0.01 and 0.19 μM. Some of the compounds are also potent affinity labels for AR. Compound 19 exhibits the highest and almost complete irreversible inhibition of AR known to date.

Full text of DOI:10.1016/S0223-5234(99)00219-6

Eur. J. Med. Chem. 34 (1999) 745−751
© 1999 Editions scientifiques et médicales Elsevier SAS. All rights reserved
745
´
Short communication
Phenylsulfonylnitromethanes as potent irreversible inhibitors of aldose reductase
Nada H. Saaba, Isaac O. Donkora, Libaniel Rodriguezb, Peter F. Kadorb, Duane D. Millera*
aDepartment of Pharmaceutical Sciences, The University of Tennessee, Memphis, TN 38163, USA
bLaboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA
(Received 27 October 1998; revised 4 March 1999; accepted 4 March 1999)
Abstract – Aldose reductase (AR) inhibition provides a viable pharmacologically direct mode for the treatment of diabetic complications. We
have synthesized a series of N-4 substituted analogues (15–21) of the known aldose reductase inhibitor phenyl-sulfonylnitromethane. The
compounds are potent inhibitors of AR with IC50s between 0.01 and 0.19 µM. Some of the compounds are also potent affinity labels for AR.
´
Compound 19 exhibits the highest and almost complete irreversible inhibition of AR known to date. © 1999 Editions scientifiques et médicales
Elsevier SAS
aldose reductase / diabetic complication / phenylsulphonyl-nitromethane / irreversible inhibitors / aldose reductase inhibitors
1. Introduction
petitive inhibition and bind to a site on the enzyme which
is independent of the substrate and NADPH cofactor
binding sites [4–6]. Using affinity-labelling studies, Ka-
dor et al. [7] demonstrated that there are three distinct
binding sites on the AR enzyme, namely, the substrate
site, cofactor site and inhibitor site. This observation is,
however, at odds with recent X-ray crystallographic
studies involving the ternary complex between AR,
NADPH and zopolrestat in which the inhibitor was found
completely sequestered into the substrate site [8]. In an
effort to study the nature of the inhibitor binding site of
AR, we previously reported a series of affinity labels
(7–10) (table I) based on the reversible inhibitor alresta-
tin [9]. Based on the remarkable irreversible inhibitory
activity of 5-iodoacetamidoalrestatin (10) in our previous
studies [7, 9] and the possibility of using affinity labels to
locate the binding site(s) of ARIs on AR, we synthesized
affinity labels (15–17) and (19–21) along with their
known acetylated derivatives (14 and 18) (table I), de-
rived from a new class (sulfonylnitromethanes) of potent
AR inhibitors, and examined the compounds for AR
inhibitory activity.
Over 50% of diabetics develop tissue-damaging com-
plications [1]. These complications develop in tissues
capable of insulin-independent glucose uptake and result
in retinopathy, nephropathy, cataract, keratopathy, neur-
opathy and angiopathy. Results of the recent Diabetes
Control and Complications Trial suggest that tight control
of blood sugar levels can reduce the incidence and
severity of diabetic complications [1]; however, on a
practical basis, tight control is difficult to maintain. This
has spurred efforts toward the development of alternative
treatments with agents acting by mechanisms indepen-
dent of the control of blood glucose. Aldose reductase
inhibitors (ARIs) have provided therapeutically useful
agents [2], which in long-term animal studies demon-
strate beneficial prevention or delay in the onset and
progression of diabetic complications with no significant
adverse effects [3]. The search for clinically useful ARIs
has been an on-going process since the late 1960s. This
effort has led to the discovery of a number of structurally
diverse compounds as ARIs (figure 1).
Kinetic studies suggest that despite their structural
variations, ARIs exhibit either uncompetitive or noncom-
2. Chemistry
Sulfonylnitromethanes have been synthesized by three
principal synthetic routes. These include the sulfonation
*Correspondence and reprints

746
Figure 1. Aldose reductase inhibitors.
of α-halo nitro compounds [10], the oxidation of α-nitro
sulfide [11] and the alkaline nitration of sulfones [12].
Alkaline nitration, which is based on a free radical chain
process rather than nucleophilic displacement of iodide
by the nitro anion, was the most fruitful method for our
synthesis. The synthesis of affinity labels 15–17 and
19–21 was achieved as depicted in figure 2. The amino
moiety of dimethylaniline (22) was protected by treat-
ment with acetic anhydride to give 23, which was reacted
with chlorosulfonic acid to yield the corresponding sul-
fonyl chloride 25. Compound 25 as well as the commer-
cially available N-acetylsulfonyl chloride (24) were re-
duced using sodium sulfite in the presence of sodium
bicarbonate buffer to give the corresponding sulfinic
acids 26 and 27 [13]. The acids were converted to their
sodium salts and then reacted with two-fold excess of
nitromethane in the presence of iodine and sodium
methoxide at 0 °C to give the corresponding N-(acetyl-
phenyl)sulfonyl nitromethane derivatives 14 and 18 [13,
14]. Basic hydrolysis of the acetyl group afforded 12 and
13. The latter compounds were treated with thiophosgene
in acetone to give the corresponding isothiocyanate
analogues 15 and 19, respectively. Treatment of 12 or 13
with chloro- or iodoacetic anhydride afforded the corre-
sponding chloro- or iodoacetamido derivatives 16 and 20
or 17 and 21, respectively.
3. Results and discussion
The sulfonylnitromethane analogues were found to be
potent inhibitors of recombinant rat lens AR with IC50s
ranging between 0.01 to 0.19 µM (table I). Subsequent
gel filtration studies for irreversible binding also indi-
cated that compounds 15–17 and 19–21 are potent affinity
labels for AR (table I). Comparison of the activities of
compounds 15–17 with that of compounds 19–21 indi-
cates that ortho dimethyl substitution potentiates irrevers-
ible inhibition of AR in this class of compounds. In
contrast to the affinity labels (7–10, table I) derived from
alrestatin, the irreversible inhibitory activity for the
present compounds is not directly linked to the chemical
reactivity of their electrophilic groups. Irreversible in-
hibitory activity for both sets of compounds decreased in
order: isothiocyanato analogues > iodoacetamido ana-
logues > chloroacetamido analogues. No correlation be-
tween reversible and irreversible inhibitory activities was
observed.
Results from mass spectrometry and molecular mod-
elling studies [7], carboxymethylation studies [15], site
directed mutagenesis studies [16], as well as kinetic
data [4–6] suggest the possibility of multiple inhibitor
binding sites on AR which are distinct from the substrate
binding site. Structurally diverse, selective affinity labels

747
Table I. Reversible (IC50) and irreversible (%) inhibition of recombinant rat lens AR.
#
R1
R2
Ra
7
8
9
NCS
NDb
56.0
0.0
46.0
89.0
NDb
0.0
NHCOCH2Cl
NHCOCH2Br
NHCOCH2I
0.55 ± 0.04
0.60 ± 0.05
0.40 ± 0.02
NDb
10
11
12
13
14
15
16
17
18
19
20
21
H
NH2
NH2
NHCOCH3
NCS
NHCOCH2Cl
NHCOCH2l
NHCOCH3
NCS
NHCOCH2Cl
NHCOCH2l
CH3
H
CH3
H
H
H
0.19 ± 0.66c
0.13 ± 0.04c
0.09 ± 0.09
0.05 ± 0.05
0.06 ± 0.07
0.09 ± 0.03
0.09 ± 0.09
0.13 ± 0.03
0.01 ± 0.04
0.03 ± 0.001
0.0
0.0
80.7
58.0
73.8
0.0
99.5
86.6
96.8
H
CH3
CH3
CH3
CH3
a
The alrestatin derivatives were previously reported [9].
ND, not determined.
Reported values from [13, 14].
b
c
can be useful tools for investigating the possibility of
multiple inhibitor sites on AR. The present results indi-
cate that compounds 19 and 21 are more potent affinity
labels than 5-iodoacetamidoalrestatin (10) and that appar-
ently complete irreversible inhibition of AR can be
achieved with compound 19. Future determination of the
exact site(s) of interaction with the enzyme by these
compounds and identification of the reactive nucleophilic
residue(s) that bind to these compounds will provide
insight into the possibility of multiple inhibitor binding
sites on AR.
reported in parts per million (δ) relative to tetramethyl-
silane (TMS) as an internal standard. Spectral data are
consistent with assigned structures. Elemental analyses
were performed by Atlantic Microlab Inc., Norcross GA,
and experimentally determined values are within ± 0.4%
of the theoretical values. Routine thin-layer chromatog-
raphy (TLC) was performed on silica gel GHIF plates
(Analtech Inc., Newark DE). Flash chromatography was
performed on silica gel (Merck, grade 60, 230–400 mesh,
60 Å). Acetonitrile (MeCN) was dried by distillation,
dimethylformamide (DMF) was dried by distillation from
P2O5. All solvents (except anhydrous MeCN and DMF)
were stored over 3 or 4 Å molecular sieves.
4. Experimental protocols
4.1.1. N-Acetyl-3,5-dimethylaniline 23
Acetic anhydride (60 mL) was added slowly to 3,5-
dimethylaniline (40 mL, 0.32 mol). The hot reaction
mixture was allowed to cool to room temperature to give
42 g (80%) of 23 as a white solid: m.p. 137–139 °C
(Lit. [13] 138 °C). IR (KBr, cm–1) 1 663 (C=O). 1H NMR
(CDCl3) δ 7.12 (s, 2H, aromatic), 6.75 (s, 1H, aromatic),
2.28 (s, 6H, 2CH3), 2.15 (s, 3H, COCH3), 1.63 (br, 1H,
NHCO).
4.1. Chemistry
Melting points were determined on a Thomas-Hoover
capillary melting point apparatus and are uncorrected.
Infrared (IR) spectra were recorded on a Perkin Elmer
System 2000 FT-IR spectrophotometer. Proton nuclear
magnetic resonance (1H NMR) spectra were recorded on
a Bruker AX 300 spectrometer. Chemical shift values are

748
Figure 2. Synthetic route to phenylsulfonylnitromethanes.

749
4.1.2. (4-Acetamido-2,6-dimethylphenyl)sulfonyl chlo-
ride 25
dissolved in dry DMF (15 mL) and cooled to 0 °C.
Nitromethane (3.22 g, 0.052 mol) was dissolved in dry
DMF (15 mL) and added slowly to the cooled NaOMe
solution. The reaction mixture was stirred at 0 °C for
15 min during which period a bright yellow precipitate
separated out. The previously prepared sodium salt of 26
was dissolved in dry DMF, cooled to 0 °C, and added to
the bright yellow mixture followed immediately by the
addition of iodine (6.08 g, 0.024 mol). The reaction was
stirred at 0 °C for 4 h and then allowed to warm to room
temperature over 30 min. The dark solution was poured
into ice-water and decolourized with sodium sulfite. It
was acidified slowly to pH 1.5 with 2 N HCl and the
resulting suspension was stored in a refrigerator over-
night followed by filtration to recover the product which
was dried and recrystallized from MeOH to give a 63%
yield of 14: m.p. 220–222 °C (dec.) (Lit. [14]
228–229 °C). IR (KBr, cm–1) 3 259 (NH), 1 674 (C=O),
1 590 (NO2), 1 553 (NO2). 1H NMR (DMSO-d6) δ 10.54
(s, 1H, NHCO), 7.90 (s, 4H, aromatic), 6.57 (s, 2H,
CH2NO2), 2.13 (s, 3H, COCH3).
In a three-necked round bottom flask fitted with a
mechanical stirrer was placed chlorosulfonic acid (100 g,
0.85 mol). The flask was cooled (12–15 °C) by means of
an ice-bath and N-acetyl-3,5-dimethylaniline (28.07 g,
0.172 mol) was added slowly over a 15 min period. After
the addition was complete, the mixture was allowed to
warm to room temperature and then was heated to 60 °C
with stirring. After 2.5 h, the mixture was added slowly to
ice-water mixture (330 mL) with stirring. The off-white
precipitate that formed was filtered and dried in a
desiccator under vacuum overnight to give 22 g of crude
product. An analytical sample was recrystallized from
benzene: m.p. 157–159 °C. IR (KBr, cm–1) 3 322 (NH),
1
1 683 (C=O), 1 360 (SO2) and 1 172 (SO2). H NMR
(CDCl3) δ 7.65 (br, 1H, NH), 7.42 (s, 2H, aromatic), 2.72
(s, 6H, 2CH3), 2.22 (s, 3H, COCH3).
4.1.3. (4-Acetamidophenyl)sulfinic acid 26
N-Acetylsulfanilyl chloride (11.9 g, 0.05 mol) was
added to a vigorously stirred solution of sodium bicar-
bonate (10 g, 0.119 mol) and sodium sulfite (14.29 g,
0.113 mol) in water (60 mL) at 70–80 °C. The mixture
was heated for 1 h and allowed to cool to room tempera-
ture. The white precipitate that separated out was redis-
solved in water and acidified with 60% sulfuric acid until
a white precipitate appeared. The suspension was left in a
refrigerator overnight. The solid was recovered by filtra-
tion and recrystallized from water to give 7.23 g (72%) of
26 as white needle-like crystals: m.p. 149–151 °C. IR
(KBr, cm–1) 3 321 (NH), 1 668 (C=O), 1 320 (SO2),
1 084 (SO2). 1HNMR (DMSO-d6) δ 10.20 (s, 1H,
NHCO), 7.73 (d, 2H, aromatic), 7.58 (d, 2H, aromatic),
2.06 (s, 3H, COCH3).
4.1.6.
(4-Acetamido-2,6-dimethylphenyl)sulfonylnitro-
methane 18
Compound 27 (2 g, 8.8 mmol) was transformed into 18
as described above for the synthesis of 14. The off-white
solid obtained was purified by column chromatography
over silica gel with hexane/ethyl acetate (1:2) as the
eluant followed by recrystallization from ethanol to yield
white crystals: m.p. 177–179 °C (Lit. [13] 179–180 °C).
IR (KBr, cm–1) 3 308 (NH), 1 677 (C=O), 1 534 (NO2).
1H NMR (acetone-d6) δ 9.50 (s, 1H, NHCO), 7.59 (s, 2H,
aromatic), 6.15 (s, 2H, CH2NO2), 2.60 (s, 6H, 2CH3),
2.05 (s, 3H, COCH3).
4.1.7. (4-Aminophenyl)sulfonylnitromethane 12
4.1.4. (4-Acetamido-2,6-dimethylphenyl)sulfinic acid 27
Sulfonylchloride 25 (22 g, 0.084 mol) was transformed
to sulfinic acid 27 as described above for the synthesis of
26. The crude product was recrystallized from water to
give 5.5 g (29%) of 27 as white crystals: m.p.
158–159 °C. IR (KBr, cm–1) 3 321 (NH), 1 668 (C=O),
A solution of 14 (0.2 g, 0.77 mmol) in 2 N NaOH
(2 mL) was heated for 1 h at 80 °C. The mixture was then
poured into ice-water mixture (10 mL) containing acetic
acid (0.3 mL) to yield a solid which was extracted with
EtOAc, dried (MgSO4), and evaporated under reduced
pressure. The residue was purified by column chromatog-
raphy over silica gel with hexane/EtOAc (1:1) as the
eluant followed by recrystallization from EtOH to give a
78% yield of 12: m.p. 128–130 °C. IR (KBr, cm–1) 3 396
(NH2), 1 554 (NO2). 1H NMR (DMSO-d6) δ 7.51 (s, 2H,
aromatic), 6.67 (s, 2H, aromatic), 6.44 (s, 2H, CH2NO2),
6.30 (s, 2H, Ar-NH2).
1
1 320 (SO2), 1 084 (SO2). H NMR (CDCl3) δ 7.31 (s,
2H, aromatic), 2.59 (s, 6H, 2CH3), 2.10 (s, 3H, COCH3).
4.1.5. (4-Acetamidophenyl)sulfonylnitromethane 14
Sodium metal (0.62 g, 0.027 mol) was dissolved in dry
MeOH and 26 (5.5 g, 0.027 mol) was added and the
mixture was stirred overnight. The solvent was removed
under reduced pressure and the residue was kept in a
desiccator. Another sample of sodium metal (1.09 g,
0.047 mol) was dissolved in dry MeOH, the solvent was
stripped off under reduced pressure, and the residue was
4.1.8. (4-Amino-2,6-dimethylphenyl)sulfonylnitromethane 13
Compound 18 (0.146 g, 0.51 mmol) was transformed
into 13 in 81% yield as described above for the synthesis
of 12: m.p. 131–133 °C (Lit. [13] 132–133 °C). IR (KBr,

750
cm–1) 3 474 (NH2), 3 376 (NH2), 1 551 (NO2). 1H NMR
(DMSO-d6) δ 7.54 (s, 2H, aromatic), 6.40 (s, 2H,
CH2NO2), 2.62 (s, 6H, 2CH3), 6.32 (s, 2H, Ar-NH2).
4.1.13. (4-Iodoacetamidophenyl)sulfonylnitromethane 17
Iodoacetic anhydride (0.106 g, 0.29 mmol) was added
to a solution of 12 (0.05 g, 0.23 mmol) in dry CH3CN
(4 mL) and the mixture was stirred at room temperature
for 18 h. The mixture was concentrated and CHCl3 was
added and kept in a refrigerator overnight to precipitate
pure 17 as pale yellow crystals in 95% yield: m.p. 165 °C.
IR (KBr, cm–1) 3 356 (NH), 1 690 (C=O). 1H NMR
(DMSO-d6) δ 10.88 (s, 1H, NHCO), 7.50 (d, 4H,
aromatic), 6.61 (s, 2H, CH2NO2), 3.87 (s, 2H, CH2I). MS
(EI, m/z) 384+. Anal. C9H9N2SO5I (C, H, N, O, S, I).
4.1.9. (4-Isothiocyanatophenyl)sulfonylnitromethane 15
Thiophosgene (12 drops) was added to a solution of 12
(0.06 g, 0.28 mmol) in dry acetone (4.5 mL) and the
mixture was stirred at room temperature for 2 h. The
solvent was removed under reduced pressure and the
crude product was purified by column chromatography
over silica gel with hexane/EtOAc (1:1) as the eluant to
yield 0.06 g (87%) of 15 as a bright yellow solid: m.p.
159–160 °C. IR (KBr, cm–1) 2 131 (NCS). 1H NMR
(acetone-d6) δ 8.09 (d, 2H, aromatic), 7.72 (d, 2H,
aromatic), 6.35 (s, 2H, CH2NO2). MS (EI, m/z) 258+.
Anal. C8H6N2S2O4 (C, H, N, O, S).
4.1.14. (4-Iodoacetamido-2,6-dimethylphenyl)sulfonyl-
nitromethane 21
This compound was synthesized as described for the
synthesis of 17 by reacting iodoacetic anhydride (0.094 g,
0.26 mmol) with 13 (0.05 g, 2.0 mmol). Compound 21
was obtained in 95% yield as white crystals. M.p. 200 °C.
IR (KBr, cm–1) 3 366 (NH), 1 723 (C=O). 1H NMR
(DMSO-d6) δ 10.68 (s, 1H, NHCO), 7.50 (s, 2H, aro-
matic), 6.49 (s, 2H, CH2NO2), 3.84 (s, 2H, CH2I), 2.51 (s,
6H, 2CH3). Anal. C11H13N2SO5I (C, H, N, O, S, I).
4.1.10. (4-Isothiocyanato-2,6-dimethylphenyl)sulfonyl-
nitromethane 19
Thiophosgene (8 drops) was added to a solution of 13
(0.06 g, 2.4 mmol) in dry acetone (3 mL) and the mixture
was stirred at room temperature for 2 h. The solvent was
removed under reduced pressure and the crude product
was purified by column chromatography over silica gel
with hexane/EtOAc (1:1) as the eluant to yield 0.065 g
(93%) of 19 as a white solid: m.p. 110–112 °C. IR (KBr,
cm–1) 2 009 (NCS). 1H NMR (acetone-d6) δ 7.35 (s, 2H,
aromatic), 6.27 (s, 2H, CH2NO2), 2.68 (s, 6H, 2CH3). MS
(EI, m/z) 286+. Anal. C10H10N2S2O4 (C, H, N, O, S).
4.2. Biological studies
4.2.1. Enzyme purification
Recombinant rat lens AR was purified by a series of
chromatographic procedures as previously de-
scribed [17]. Briefly, AR was released from E. coli by
sonication and the mixture was centrifuged at 10 000 g
for 10 min. The supernatant was then subjected to gel
filtration on a Sephadex G-75 column (2.5 × 90 cm),
equilibrated with 10 mM imidazole-HCl buffer, pH 7.5
containing 7 mM 2-mercaptoethanol and eluted with the
same imidazole buffer. The eluent was collected in
220-drop aliquots (ca. 10 mL) and fractions containing
AR activity were applied to a Matrex Gel Orange A
affinity column (2.5 × 15 cm). The affinity column was
washed with the imidazole buffer (ca. 500 mL) and the
enzyme was eluted with the same imidazole buffer
containing 0.1 mM NADPH. Fractions eluted with
NADPH were chromatofocused on a Mono P (HR 5/20)
column developed at a flow rate of 1 mL/min with
Polybuffer 74 (diluted 10-fold and containing 7 mM
2-mercaptoethanol). The protein concentration of the
eluent was monitored at 280 nm and peaks containing AR
activity were collected and concentrated on Centricon 10
filters.
4.1.11.
(4-Chloroacetamidophenyl)sulfonylnitro-
methane 16
Chloroacetic anhydride (73 mg, 0.43 mmol) was added
to a solution of 12 (0.06 g, 0.27 mmol) in dry CH3CN
(4 mL) and the mixture was stirred at room temperature
for 18 h. The solvent was concentrated to precipitate pure
16 in 0.044 g (55%) yield as a white solid: m.p. 212 °C.
IR (KBr, cm–1) 3 279 (NH), 1 684 (C=O). 1H NMR
(DMSO-d6) δ 10.86 (s, 1H, NHCO), 7.92 (d, 4H,
aromatic), 6.60 (s, 2H, CH2NO2), 4.34 (s, 2H, CH2Cl).
MS (EI, m/z) 292 +. Anal. C9H9N2SO5Cl (C, H, N, O, S,
Cl).
4.1.12. (4-Chloroacetamido-2,6-dimethylphenyl)sulfonyl-
nitromethane 20
Compound 13 (0.05 g, 2.0 mmol) was treated as de-
scribed for the synthesis of 16 to yield 0.06 g (87%) of 20
as a bright yellow solid: m.p. 160–161 °C. IR (KBr,
cm–1) 3 366 (NH), 1 723 (C=O). 1H NMR (DMSO-d6) δ
10.66 (s, 1H, NHCO), 7.53 (s, 2H, aromatic), 6.50 (s, 2H,
CH2NO2), 4.31 (s, 2H, CH2Cl), 2.56 (s, 6H, 2CH3). MS
(EI, m/z) 320+. Anal. C11H13N2SO5Cl (C, H, N, O, S, Cl).
4.2.2. Enzyme assay
Reductase activity was spectrophotometrically assayed
on a Shimadzu UV 2100U spectrophotometer by follow-
ing the decrease in the absorption of NADPH at 340 nm

751
over a 4-min period with DL-glyceraldehyde as sub-
strate [9]. Each 1 mL cuvette contained equal units of
enzyme, 0.10 M Na, K phosphate buffer, pH 6.2, 0.3 mM
NADPH with/without 10 mM substrate and inhibitor.
Appropriate controls were employed to negate potential
changes in the absorption of nucleotide and/or protein
modification reagents or ARIs at 340 nm in the absence of
substrate.
References
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ethanol which stabilizes AR [9]. Equal aliquots of en-
zyme were then combined with either reversible or
irreversible inhibitor dissolved in 0.1 M sodium phos-
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proceed at room temperature for 15 min. Reversible and
unreacted inhibitor were then removed from the protein
by gel filtration with a NAP-5 desalting column with
0.1 M phosphate buffer, pH 7.0, containing 10 mM
2-mercaptoethanol. Residual AR activity was spectropho-
tometrically measured. All experiments were conducted
at least in triplicate.
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Abdel-Ghany Y.S., Miller D.D., Bioorg. Med. Chem. 3 (1995)
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Acknowledgements
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Biol. Chem. 266 (1991) 24031–24037.
This work was supported in part by NIH grant 1 R15
EY0936-02 (I.O.D). We thank Mr John Miller for run-
ning the Mass Spectra of the compounds.
[17] Old S.E., Sato S., Kador P.F., Carper D.A., Proc. Natl. Acad. Sci.
USA 87 (1990) 4942–4945.