Halogenated 2,5-pyrrolidinediones: Synthesis, bacterial mutagenicity in Ames tester strain TA-100 and semi-empirical molecular orbital calculations

Article abstract of DOI:10.1016/S1383-5718(00)00129-7

The chloroimide 3,3-dichloro-4-(dichloromethylene)-2,5-pyrrolidinedione, a tetrachloroitaconimide, is the principal mutagen produced by chlorination of simulated poultry chiller water. It is the second most potent mutagenic disinfection by-product of chlorination ever reported. Six of seven new synthetic analogs of this compound are direct-acting mutagens in Ames tester strain TA-100. Computed energies of the lowest unoccupied molecular orbital (E_LUMO) and of the radical anion stability (ΔH_f/^rad - ΔH_f) from MNDO-PM3 for the chloroimides show a quantitative correlation with the Ames TA-100 bacterial mutagenicity values. The molar mutagenicities of these direct acting mutagenic imides having an exocyclic double bond fit the same linear correlation (ln_Mm vs. E_LUMO; ln_Mm vs. ΔH_f/^rad - ΔH_f) as the chlorinated 2(5H)-furanones, including the potent mutagen MX, 3-chloro-4-(dichloro-methyl)-5-hydroxy-2(5H)-furanone, a by-product of water chlorination and paper bleaching with chlorine. Mutagenicity data for related haloimides having endocyclic double bonds are also given. For the same number of chlorine atoms, the imides with endocyclic double bonds have significantly higher Ames mutagenicity compared to their structural analogs with exocyclic double bonds, but do not follow the same E_LUMO or ΔH_f/^rad - ΔH_f correlation as the exocyclic chloroimides and the chlorinated 2(5H)-furanones.

Full text of DOI:10.1016/S1383-5718(00)00129-7

Mutation Research 490 (2001) 89–98
Halogenated 2,5-pyrrolidinediones:
synthesis, bacterial mutagenicity in Ames tester strain
TA-100 and semi-empirical molecular orbital calculations
Beverly A. Freeman, Robert E. Wilson, Ronald G. Binder, William F. Haddon∗
US Department of Agriculture, Western Regional Research Center, Agricultural Research Service,
800 Buchanan Street, Albany, CA 94710, USA
Received 12 February 2000; received in revised form 29 August 2000; accepted 31 August 2000
Abstract
The chloroimide 3,3-dichloro-4-(dichloromethylene)-2,5-pyrrolidinedione, a tetrachloroitaconimide, is the principal muta-
gen produced by chlorination of simulated poultry chiller water. It is the second most potent mutagenic disinfection by-product
of chlorination ever reported. Six of seven new synthetic analogs of this compound are direct-acting mutagens in Ames tester
strain TA-100. Computed energies of the lowest unoccupied molecular orbital (ELUMO) and of the radical anion stability
(1Hfrad − 1Hf ) from MNDO-PM3 for the chloroimides show a quantitative correlation with the Ames TA-100 bacterial
mutagenicity values. The molar mutagenicities of these direct acting mutagenic imides having an exocyclic double bond fit
the same linear correlation (ln Mm vs. ELUMO; ln Mm vs. 1Hfrad − 1Hf ) as the chlorinated 2(5H)-furanones, including the
potent mutagen MX, 3-chloro-4-(dichloro-methyl)-5-hydroxy-2(5H)-furanone, a by-product of water chlorination and paper
bleaching with chlorine. Mutagenicity data for related haloimides having endocyclic double bonds are also given. For the same
number of chlorine atoms, the imides with endocyclic double bonds have significantly higher Ames mutagenicity compared
to their structural analogs with exocyclic double bonds, but do not follow the same ELUMO or 1Hfrad − 1Hf correlation as
the exocyclic chloroimides and the chlorinated 2(5H)-furanones. Published by Elsevier Science B.V.
Keywords: Chlorination; Mutagenicity; Pyrrolidinediones; Chloroimides; MX; Computation; Semi-empirical; LUMO
1. Introduction
foods by reducing the levels of human bacterial
pathogens. However, excessive chlorination produces
measurable Ames mutagenicity in the processing
waters when the levels of chlorination significantly
exceed the amounts established for use in poultry
processing plants [2]. Recycling of these mutagenic
by-products could limit the reuse of water in com-
mercial food processing. The objectives of our study
of disinfection by-products from poultry chlorination
were to establish the structure of the active compounds
and to assess their contribution to Ames mutagenicity.
To elucidate chemical structures of the poultry
chiller water mutagens, we carried out measure-
The chlorine disinfection by-product 3,3-dichloro-4-
(dichloromethylene)-2,5-pyrrolidinedione (1c, Fig. 1)
is the second most potent direct acting Ames mutagen
ever reported in studies of chlorination [1]. We deter-
mined the structure of the new mutagenic chloroimide
(1c), in the course of studying the Ames mutagenic-
ity produced by chlorination of chiller water used in
poultry processing operations [1–3]; chlorination of
poultry chiller water improves the safety of processed
∗ Corresponding author.
1383-5718/01/$ – see front matter Published by Elsevier Science B.V.
PII: S1383-5718(00)00129-7

90
B.A. Freeman et al. / Mutation Research 490 (2001) 89–98
is the most potent mutagenic chlorine disinfection
product ever reported. The molar mutagenicity of the
direct-acting mutagen (1c) is 1450 rev/nmol in tester
strain TA-100 without microsomal activation [1].
For strong electrophiles such as MX and the
chloroimides, it has been difficult to isolate specific
reaction products with nucleophilic reagents [9–11].
Early observations of DNA strand breaks caused by
MX [12] have been supplemented by more recent
studies showing that such reactions may be complex at
the molecular level [13,14]. The bacterial mutagenic-
ity of the MX family of compounds can be eliminated
or reduced by reaction with nucleophilic reagents
[9–11]. The mutagenicity produced by 1c in poultry
chiller water can be rapidly eliminated by l-cysteine,
N-acetyl-l-cysteine and other common nucleophilic
reagents [15] added to poultry chiller water.
MX and the mutagenic tetrachloroimide from
our simulated food processing mixture are similar
in chemical structure in that both are chlorinated
five-membered ring heterocycles (2a vs. 1c). Thus it
was of interest to explore the possible similarity of
structure-activity effects on Ames mutagenicity for
the chlorofuranones [16–21] and the chloroimides.
In this work, we attempt to link the toxicology of
these two different classes of electrophilic compounds
with the demonstration that calculated energies of
the LUMOs and the calculated radical ion heats of
formation (1Hfrad − 1Hf) yield similar correla-
tions with observed Ames mutagenicity. ELUMO and
1Hfrad − 1Hf were chosen because they are likely
indicators of electrophilic reactivity. We include syn-
thetic methods, mutagenicity data and computational
results for chloroimides having both endo- and exo-
cyclic double bonds; all the known MX analogs have
endocyclic double bonds only. Open-ring analogs of
MX make minor contributions to the total Ames mu-
tagenicity of chlorinated drinking water [22]. There
was no evidence in our studies of poultry chlorina-
tion for additional imides, or for the occurrence of
open-ring analogs [1].
Fig. 1. Structures of mutagenic chloroimides (1a–h) and chloro-
furanones (2a–j).
ments on a simulated processing mixture at a high
chlorination level. Compound 1c accounted for a
substantial fraction of the observed mutagenicity at
1500 ppm chlorination [1]. This single compound
result had precedent in the previous isolation of
3-chloro-4-(dichloro-methyl)-5-hydroxy-2(5H)-fura-
none, MX, 2a in Fig. 1, which accounted for most of
the observed Ames mutagenicity in the Kraft paper
bleaching process and in the chlorination of drinking
water in contact with humic material. MX was first
identified by Holmbolm [4,5], and has recently been
shown to be carcinogenic in rats [6]. MX is of consid-
erable interest because of its demonstrated presence
in drinking water supplies [7] and the still unresolved
question of its possible importance in human health
[8]. MX, with a molar mutagenicity in the range
2600–10 000 revertants per nanomole in TA-100,
2. Materials and methods
2.1. Chemical synthesis
The synthesis of four chlorinated 2,5-pyrrolidine-
diones having exocyclic double bonds was reported

B.A. Freeman et al. / Mutation Research 490 (2001) 89–98
91
previously [1]. For the current study, three additional
chloroimides and a single mixed bromo-chloroimide
having an endocyclic double bond (Fig. 1, 1e–h) were
synthesized. Three of the four newly-synthesized com-
pounds are direct-acting mutagens in TA-100.
to tetramethylsilane as internal standard (δ = 0.0
ppm).
Preparation of 3-chloro-4-dichloromethyl-1H-
pyrrole-2,5-dione (1e). Citraconimide was first pre-
pared as previously described [1,23]. The mixture
obtained from a four-day chlorination of citracon-
imide was distilled under vacuum with bath tempera-
ture to 220◦C. The residue was dissolved in toluene,
treated with activated carbon, filtered through sil-
ica gel, then re-distilled with bath temperature to
160◦C. The major component, RI 1551 by gas chro-
matography, was concentrated by silica gel chro-
matography and finally separated from an impurity
3,3-dichloro-4-dichloromethylene-2,5-pyrrolidinedi-
one, by chromatography on a silica gel column with
eluting solvent 2% ether in hexane. Crystals formed
in the neat material after a week. The solid was re-
crystallized from CCl4 to give 1e, m.p.: 70.0–70.7◦C
(MS: m/z 215 (24), 213 (26) (M+), 180 (26), 178 (39),
Compounds to be chlorinated were added to a solu-
tion of chlorine in CCl4 and exposed to direct sunlight
for one to eight days, usually producing a complex
mixture of compounds. Typically, 10–15% excess
chlorine was present initially; compounds of low
solubility were first dissolved in trifluoroacetic acid.
Purifications of the reaction mixtures were carried
out by silica gel chromatography using a specially
prepared phenyl silica packing. The column packing
was prepared by heating 60 g silica gel 60 H, dried at
175◦C, in 220 ml benzyl alcohol in a bomb for 3 h at
200◦C; product was thoroughly rinsed with acetone
and ether and oven-dried to give 67.2 g packing. This
product was further treated with 15 ml hexamethyldis-
ilazane in boiling benzene. After cleaning and drying,
67.4 g of packing was obtained. Use of this packing
facilitated separation of chloroimide products from
less polar artifacts present in the reaction mixtures.
Analytical HPLC analyses utilized a 4.6 mm ×
25 cm phenyl column (Microsorb-MV, Rainin Instru-
ment Co.) and normal phase chromatography: hexane
to 2% isopropanol in 8 min, then to 4% isopropanol
in 8 min, then held at 4% isopropanol at 13 min,
all at 1 ml/min. GC-MS analyses were carried out
on a VG 7070/HS mass spectrometer (Micromass,
Manchester, UK) and HP 5830 gas chromatograph
(Hewlett-Packard, Palo Alto, CA) or on a Finnigan
GCQ ion trap mass spectrometer (Thermosepara-
tions, San Jose, CA) using 0.25 mm i.d., 0.25 ␮m,
30 m DB-5 or DB-5MS (5% phenyl, 95% methyl sil-
icone) capillary columns (J & W Scientific, Folsom,
CA) with splitless injection. Electron ionization (EI)
spectra were recorded at 70 eV ionizing voltage. Typ-
ically, GC runs used linear temperature programs of
3◦C/min from 60 to 280◦C/min, and splitless injection
at 220◦C. The ion source temperature was 180◦C and
transfer lines to the mass spectrometer were main-
tained at 230◦C. Retention index (RI) values were
assigned by linear interpolation between the reten-
tion times of n-alkane standards. Proton (400 MHz)
and 13C NMR (100 MHz) spectra were determined
in CDCl3 solutions on a Bruker ARX400 spectrom-
eter. Chemical shift values (δ) are in ppm relative
1
170 (11), 160 (13), 109 (59), 107 (100). H NMR:
δ6.61 (s, 2H), 7.79 (br s, 1H); 13C NMR: δ164.33,
162.77, 137.34, 135.90, 58.02; UV: λmax, 232 nm).
Preparation of 3-chloro-4-chloromethyl-1H-pyrrole-
2,5-dione (1f ). Chloromethyl maleic anhydride, pre-
pared by the procedure of Schreiber et al. [24], was
chlorinated, yielding 3-chloromethyl-3,4-dichloro-
succinic anhydride, which was twice distilled under
vacuum to produce 3-chloro-4-chloromethylmaleic
anhydride (RI 1206; MS: m/z 182 (24), 180 (35)
(M+), 147 (23), 145 (68), 138 (24), 136 (38), 101
(54), 73 (100); 1H NMR: δ4.40 (s); 13C NMR: δ30.90,
138.26, 139.37, 158.70, 160.67). Stirring the anhy-
dride in a mixture of urea and salt at 130◦C converted
it to the imide [22], which was purified by silica gel
chromatography and recrystallized several times from
CCl4 to give 1f, m.p.: 98.2–98.8◦C (MS: m/z 181
(52), 179 (83) (M+), 161 (36), 146 (32), 144 (98),
136 (41), 73 (100). 1H NMR: δ4.36 (s, 2H), 7.82
(s, 1H); 13C NMR: δ166.28, 163.55, 138.30, 136.49,
31.16; UV: λmax, 230 nm).
Preparation of 3-chloro-4-methyl-1H-pyrrole-2,5-
dione (1g). Compound 1g was prepared from citra-
conic anhydride (Aldrich Chemicals, 99% pure by
GC analysis) by the procedure of Earl et al. [23] (MS:
m/z 147 (32), 145 (100) (M+), 102 (19), 74 (17), 64
(47), 39 (39); UV: λmax, 230 nm).
Preparation of 3-bromomethyl-4-choromaleimide
(1h). Compound 1g (1.2 g), was combined with

92
B.A. Freeman et al. / Mutation Research 490 (2001) 89–98
trifluoroacetic acid (2 ml), and bromine (1 ml) and
then exposed to sunlight for seven days. After re-
moval of the acid and bromine by evaporation, the
resulting residue was chromatographed on silica gel
with eluting solvents CCl4, benzene and ether. The
diethyl ether eluate contained 1h (RI = 1547) and
3,4-dibromo-3-chloro-4-methylprrolidindione (RI =
1664). 1h was crystallized from CCl4 to an estimated
purity (GC) of 97%. 1H NMR (400 MHz) δ 4.20
(s, CH2, 2H), 7.93 (broad s, NH, 1H); 13C NMR
2.3. Computational methods
Molecular modeling calculations utilized an
INSIGHT II integrated software package (Biosym-
MSI) with a Silicon Graphics Indigo workstation. The
initial structures prepared using the Sketcher module
were submitted to preliminary geometry optimiza-
tion using molecular mechanics methods with the
INSIGHT II DISCOVER interface in the BUILDER
module. Following these initial approximations of
structure, semi-empirical molecular orbital and 1Hf
calculations were performed with MOPAC 6.0 using
the MNDO-PM3 parameterization set. All molecules
were optimized using the default BFGS minimiza-
tion type. Peptide corrections had no effect on the
calculations. The MOPAC keyword PRECISE for ter-
mination of the calculations was set 100-fold below
the default value as in previous work [17]. The gra-
dient norm was 5 and the shift level was 15. Pulay’s
convergence method and 200 self-consistent field
iterations were utilized in the calculations. The gradi-
ent type was NLLSQ. For all cases, the Herbert’s or
Peter’s test was satisfied in BFGS.
==
==
(100 MHz) δ 166.27 (C O), 163.66 (C O), 137.08
(C–Cl), 136.99 (C–CH2Br), 15.36 (CH2Br).
Preparation of 3-chloro-4-(dichloromethyl)-5-
hydroxy-2(5H)-furanone (MX, 2a). We followed the
method of Padmapriya [24]. Final purification of prod-
uct was on silica gel H pre-washed with hydrochloric
acid, then rinsed and dried.
Caution. The compounds reported in this study, with
the exception of 1g, are mutagenic in the Ames mu-
tagenicity assay using Salmonella typhimurium strain
TA-100 without microsomal activation. Therefore cau-
tion should be exercised in their use and disposal.
To avoid trapping a molecule with rotatable groups
in a local minimum, calculations were carried out for
each 10◦ increment of bond rotation when appropri-
ate using the Torsion command in INSIGHT II. Each
structure so obtained was submitted to MOPAC and
the confomer having the lowest 1Hf was taken as
the most stable structure. For calculations on neutral
molecules, the lowest electronic state was used and the
charge was zero. For calculations on radical anions,
the doublet electronic state was used and the charge
was −1.
2.2. Mutagenisis assay
Haloimide samples were dissolved in 1,4-dioxane
and tested for mutagenicity in the Ames/Salmonella
assay using the standard plate incorporation method
[25]. For MX, measurements were carried out in
both DMSO and in peroxide-free 1,4-dioxane, which
gave the same result within statistical error (data not
shown). All measurements were conducted in S. ty-
phimurium tester strain TA-100 without activation by
rat liver homogenate fraction S9. Mutagenicity was
calculated from the linear portion of the dose-response
curves using three to five concentrations in the linear
range of the assay, which was determined by pre-
liminary range-finding experiments. Positive control
plates contained 0.4 ␮l per plate of methyl methane
sulfonate. Mean molar mutagenicity values (Mm),
expressed as revertants per nanomole, were calcu-
lated from the slopes of the dose-response curves as
determined by linear regression. Duplicate plating
was used to determine the dose-response curves for
all compounds. Compound 1g was non-mutagenic,
exhibiting less than twice the number of background
revertants [25], as shown in Table 1.
3. Results and discussion
3.1. Mutagenicity
In addition to performing the standard tests for
evaluating the activity of the TA-100 tester strain used
in our work, we directly compared the response of this
strain to MX independently synthesized in our labo-
ratory. Our measured value is 7825 rev/nmol for the
bacterial mutagenicity of MX in TA-100, without S9
activation, as shown in Table 1. This value falls within
the range reported in the literature of about 2800 to

B.A. Freeman et al. / Mutation Research 490 (2001) 89–98
93
Table 1
Ames mutagenicity measurements in S. typhimurium strain TA-100a
Compound
MX
Dose (␮g/plate) (nmol/plate)b
Rev/plate
Mm (slope(rev/nmol))c
7825
r2
0.0200 (0.092)
0.0100 (0.046)
0.0075 (0.034)
0.0050 (0.023)
0.0025 (0.011)
(0)
988; 876
749; 654
495; 654
398; 402
336; 349
232; 248; 204; 220
0.966
Solvent controld
490
0.981
1e
0.500 (2.43)
0.250 (1.16)
0.125 (0.58)
0.050 (0.23)
(0)
1402; 1238
655; 589
379; 366
232; 248; 204; 220
199; 215
Solvent control
1f
0.400 (2.22)
0.300 (1.67)
0.200 (1.11)
0.100 (0.56)
0
1249; 1224
801; 893
640; 576
348; 379
195; 213
459
0.975
Solvent control
1g
200 (1380)
150 (1035)
100 (690)
50
Kill
Kill
223; 198
179; 179
195; 213
<0.3
545
Solvent control
0
1h
0.500 (2.23)
0.250 (1.11)
0.100 (0.45)
(0)
1373; 1449
753; 785
412; 422
184; 219
0.999
Solvent control
a All measurements in 1,4-dioxane. The positive control, methyl methane sulfonate, gave >1200 rev/plate in all assays at 0.4 ␮g/plate.
b Mm, molar mutagenicity, calculated by multiplying the dosage (␮g/plate) by 1000 and dividing by the molecular weight of the
compounds in g/mol.
c Slope in rev/nmol, by linear regression.
d Background counts above 200 are typical for use of 1,4-dioxane as solvent (B.N. Ames laboratory, personal communication).
above 10 000 rev/nmol [16,18]. Thus in their attempts
to correlate mutagenicity values with computed prop-
erties, LaLonde et al. [17] used 4000 rev/nmol as
the mutagenicity of MX, and Tuppurainen et al.
[18], used a value of 6300 rev/nmol for MX. Be-
cause our measured value for MX fell in the range
of literature values, no adjustments were made for
comparisons of chloroimide mutagenicities with data
obtained elsewhere for MX and its various analogs.
Furthermore, there was no solvent dependency for
the bacterial mutagenicity of MX in TA-100, but for
the chloroimides, mutagenicities measured in DMSO
or ethanol solutions were significantly lower than in
1,4-dioxane solution; 800 rev/nmol for 1c in ethanol
vs. about 1500 rev/nmol in 1,4-dioxane [1]; thus all
TA-100 mutagenicity data reported in this work is
based on measurements carried out in 1,4-dioxane.
The 1,4-dioxane produces higher background counts
than DMSO, but as indicated in the table, the values
are well within an acceptable range [25].
The structures of the haloimide compounds are
shown in Fig. 1. For the computational study we
synthesized four novel five-carbon haloimides hav-
ing structures containing endocyclic double bonds
(1e–h). The imide containing a single chlorine (1g)
was non-mutagenic in TA-100 by the criteria of Pri-
val and Dunkle [26], but the imides with two and
three chlorines (1e and 1f), and the single mixed
bromo, dichloro compound (1h) were all strongly
mutagenic. They exhibited responses in TA-100 of

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B.A. Freeman et al. / Mutation Research 490 (2001) 89–98
Table 2
Computed molecular properties and Ames mutagenicity values for chloroimides and chlorofuranones
1Hfrad − 1Hf
This workb
a
Compound
Mm (rev/nmol)
(ln Mm)
Reference
ELUMO (eV)
This workb
[17]
[18]
[17]
[18]
1a
1b
1c
1d
1e
1f
1g
1h
MX, 2a
MX, 2a
MX, 2a
2b
2c
2d
2e
2f
2g
2h
0.24
7.7
1450
28
490
459
<0.3c
545
7825
3840
6298
177
−1.427
2.041
7.279
3.332
6.194
[1]
[1]
[1]
[1]
(b)
(b)
(b)
(b)
−1.00
−1.21
−1.36
−1.59
−1.85
−1.69
−1.41
−1.89
−1.46
−38.284
−44.246
−47.736
−50.882
−58.513
−53.790
−45.70
6.129
<−1.20
6.301
−59.604
−50.55
8.965
8.253
8.748
5.176
6.361
1.593
1.353
−1.184
−0.749
−0.906
1.979
(b)
−1.53
−1.51
−52.7
−54.19
[17]
[18]
[20]
[20]
[20]
[20]
[20]
[16]
[17]
[21]
[21]
−1.32
−1.30
−1.05
−1.11
−0.85
−0.90
−0.93
−1.15
−0.93
−1.32
−1.31
−1.08
−1.13
−0.90
−0.91
−0.93
−0.97
−0.93
−1.29
−1.32
−1.09
−1.08
−0.85
−0.91
−47.16
−47.83
−41.44
−42.22
−37.54
−34.38
−38.82
−40.54
−34.34
−47.2
−47.6
−41.5
−43.4
−37.5
−34.4
−35.8
−41.9
−34.4
−48.15
−49.46
−43.49
−43.23
−37.13
−39.04
579
4.920
3.870
0.306
0.473
0.404
7.24
2I
2j
−1.24
−1.02
−46.70
−40.59
0.171
−1.766
a Molecular mutagenicity in Ames tester strain TA-100 without metabolic activation.
b This work, see Table 1.
c Non-mutagenic, see Table 1.
about 500 rev/nmol, as shown in Table 1. Table 2
summarizes our previously measured values of muta-
genicity for the three exocyclic chloroimides (1a–c),
the four-carbon compound (1d), and the previously
reported values for the halofuranones (2a–j). Fig. 1
shows the structures of the chlorofuranones which we
have used for comparison with our mutagenicity and
computational data for 1a–h.
For the tri-chloroimide isomers 1e (endocyclic
double-bonded) vs. 1b (exocyclic double-bonded),
both with two chlorines on the exocyclic carbon,
the increase in mutagenicity for the internal vs. ex-
ternal double bond is a factor of about 60. In like
manner the dichloroimide with an endocyclic dou-
ble bond 1f (459 rev/nmol) has about 1900 times the
mutagenicity of 1a (0.24 rev/nmol), although in this
case the exocyclic carbon has 1 and 2 bonded chlo-
rines, respectively. The increased mutagenicity for the
chloroimides which have an endocyclic double bond
would be consistent with the localization of positive
charge at C-4, the likely site for a 1,4-Michael addition
reaction. A positive charge at C-4 would be inductively
stabilized by the exocyclic mono- or dichloromethyl
group. For the chloroimides with exocyclic double
bonds, the Michael addition site would more likely be
the exocyclic carbon, where directly-bonded chlorine
atoms would be less stabilizing because of resonance.
The relatively greater difference in mutagenicity be-
tween 1f vs. 1a is consistent with the location of two
vs. one chlorine on the exocyclic carbon for 1a. In
1f both chlorines may inductively stabilize the pos-
itive charge at C-4, whereas for 1a, with charge on
C-6, there is no inductive stabilization by the two
chlorines.
Interestingly, the tetrachloroimide (1c), which has
the highest mutagenicity of all the chloroimides, is
the only detectable product of chlorination in the
simulated food processing mixture that we exam-
ined. It was not possible to synthesize a 4-chlorine
imide with internal double bonds. However, the
increase in mutagenicity between the di-chloro to
tri-chloro compounds 1e and 1f is very small, sug-
gesting that addition of a fourth chlorine should
have little effect. Therefore 1c appears to be the

B.A. Freeman et al. / Mutation Research 490 (2001) 89–98
95
most potent mutagen for the chloroimide series
of compounds. This conclusion is consistent with
the mutagenicities of the chlorohydroxyfuranones,
where the increase in mutagenicity between the
mono- and dichlorohydroxyfuranones (2g, 2c) is
large, a factor of 1200, but the relative increase
from addition of a third chlorine to give MX (2a)
is only a factor of 6.6. The bromine-containing
imide (1h), had mutagenicity comparable to its
structural analog (1f). This result is consistent
with similar studies reported recently for mixed
halogen-substituted 4-methyl-2(5H) furanones by
LaLonde [27].
order of 0.1 eV or less except for compound 2i, where
the difference was −0.18 and 0.09 eV, respectively.
The computed differential negative ion radical heats
of formation agreed well with LaLonde’s reported
values, the maximum deviation being 1.4 Kcal/mol for
2i; however, much larger deviations, up to 6.2 Kcal,
were observed from Tuppurainen’s calculations of
(1Hfrad − 1Hf). We did not re-measure the muta-
genicities for any of the furanone compounds except
MX.
Fig. 2a shows the ELUMO values plotted against
the mutagenicity for ten chlorofuranones and the six
chloroimides, including the compound in our study
containing bromine (1h). Notably, the ELUMO val-
ues for both the chlorofuranones and the chloroimides
with exocyclic double bonds are well-correlated. The
least squares fit to the combined set of data for the 13
chlorofuranones and chloroimides with external dou-
ble bonds gives the following equation:
3.2. Computational results
There is good evidence that the halogenated imides
may behave chemically as electrophiles from previ-
ous studies of the chemistry of citraconimide [23],
and 1c [15]. Success by others in relating the po-
tency of Ames mutagens to computed molecular prop-
erties has been based on the idea that the energy of
the lowest unoccupied molecular orbital (ELUMO), and
the electron affinity (1Hfrad − 1Hf) serve a mea-
sure of the reactivity toward nucleophilic reagents.
We have therefore investigated the calculated ELUMO
and radical anion heats of formation (1Hfrad − 1Hf)
as indicators of mutagenicity for this new series of
compounds.
ln Mm = −17.10ELUMO − 17.01 (r2 = 0.921) (1)
which is plotted in Fig. 2a. If we exclude the val-
ues derived from the chlroimides and use only the
re-calculated values of ELUMO for the chlorofura-
nones, the equation for the least squares line is
ln Mm = −16.1ELUMO − 16.5 (r2 = 0.953)
(2)
Semi-empirical computations using MNDO-PM3
gave satisfactory calculations of both the LUMO
energy and (1Hfrad − 1Hf) in all cases, yielding
MOPAC-derived bond distances and angles which
were in excellent agreement with those obtained by
X-ray crystallography for 1b and 1c [1] (data not
shown). The computed structures are essentially pla-
nar for both compounds, in agreement with the X-ray
crystallography results.
Table 2 summarizes the computed values of LUMO,
(1Hfrad − 1Hf) and measured mutagenic response
in TA-100 of all compounds reported in this study.
Comparisons with literature values of ELUMO and
(1Hfrad − 1Hf) are included. For the thirteen struc-
tures taken from previous work, we re-computed the
LUMO energies and radical anion heats of formation
using the Biosym INSIGHT II software package. For
the LUMO energy values, the deviations from pub-
lished data of LaLonde and Tuppurainen are on the
This second equation differs slightly from that orig-
inally given by LaLonde, ln Mm = −14.97ELUMO
−
14.37 (r2 = 0.925) [17] because we have used
re-calculated values of ELUMO using our software
(see Table 2).
Using Eq. (1), the predicted TA-100 response for the
chloroimides 1a–c is 1.1, 39.7 and 516 rev/nmol, re-
spectively. The calculations give a good semi-quanti-
tative prediction of the measured mutagenic potencies
of 0.24, 7.7 and 1450 rev/nmol obtained in our labo-
ratory for these compounds (Table 2).
The calculated negative anion radical heat of
formation data gave comparable results. The least
squares fit to data consisting of the recalculated,
(1Hfrad − 1Hf) values for the ten chlorofuranones
and the chloroimides with exocyclic double bonds
gives the following equation, again using the pub-
lished data for mutagenicities of the chlorofuranones
shown in Table 2.

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B.A. Freeman et al. / Mutation Research 490 (2001) 89–98
Fig. 2. (a) Plot of energy of lowest unoccupied molecular orbital, LUMO, computed from MNDO-PM3, for chloroimides and chlorofuranones
against natural log of mutagenicity in Ames tester strain TA-100 without microsomal activation. (b) Plot of radical ion stability vs. natural
log of mutagenicity in TA-100. Least squares lines include all compounds except the chloroimides with internal double bonds (1d–h).
ln Mm = −0.633(1Hfrad − 1Hf) − 24.39
(r2 = 0.903)
double bonds. For the chlorofuranones only, but using
our recalculated values for (1Hfrad −1Hf), we obtain
the following:
(3)
which is plotted in Fig. 2b. Eq. (3) predicts the mu-
tagenicity of 1a–c to be 0.85, 37.3 and 339 rev/nmol,
respectively, again in reasonable agreement with the
measured values for the chloroimides with exocyclic
ln Mm = −0.602(1Hfrad − 1Hf) − 22.96
(r2 = 0.930)
(4)

B.A. Freeman et al. / Mutation Research 490 (2001) 89–98
97
also in good agreement with the published equation
Acknowledgements
of LaLonde, et al. [17], ln Mm = −0.538(1Hfrad
−
1Hf) − 20.38 (r2 = 0.949).
The authors thank Leslie A. Harden for carrying out
mass spectral measurements, and acknowledge helpful
discussions with Rosalind Y. Wong, Robert E. Lundin
and Koni Stone.
The correlation of ELUMO and (1Hfrad − 1Hf)
with mutagenicity from Eqs. (1) and (3) for the
chloroimides with exocyclic double bonds and
the chlorofuranone derivatives would significantly
over-predict mutagenicity if applied directly to the
endocyclic imides 1e–h. Dichloromaleimide (1d)
with four carbons, also lies below the line estab-
lished by the exocyclic imides and the chlorofur-
nanones. The published chorofuranone data include
one four-carbon compound, mucochloric acid (2i)
which fits the same line as the five-carbon chlorofu-
ranones for both ELUMO and (1Hfrad − 1Hf) [17].
The fact that this four-carbon chlorofuranone fits
the same equation as the five-carbon analogs sug-
gests that double bond position may be the important
structural parameter in establishing the correlation.
In any case, the endocyclic chloroimides appear
to follow a different LUMO-mutagencity relation-
ship than their counterparts with exocyclic double
bonds.
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