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Do carboxylic/sulfonic acid halides really present a mutagenic and carcinogenic risk as impurities in final drug products? Alexander Amberg, James Harvey, Andreas Czich, HansPeter Spirkl, Sharon Robinson, Angela White, and David P Elder Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.5b00106 • Publication Date (Web): 12 Jun 2015 Downloaded from http://pubs.acs.org on June 18, 2015

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Organic Process Research & Development

Do carboxylic/sulfonic acid halides really present a mutagenic and carcinogenic risk as impurities in final drug products? Authors: Alexander Amberg,*‡ James S. Harvey, Andreas Czich,‡ Hans-Peter Spirkl, ‡ Sharon Robinson, Angela White, David P. Elder  ‡

Sanofi-Aventis Deutschland GmbH, R&D DSAR / Preclinical Safety FF, Industriepark Hoechst, Bldg.

H831, D-65926 Frankfurt 

GlaxoSmithKline Pre-Clinical Development, Park Road, Ware, Hertfordshire SG12 0DP, UK

* Corresponding Author: E-mail: [email protected]

ACS Paragon Plus Environment


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TOC Graphic


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Abstract There is substantial mutagenicity data on acyl/sulfonyl halides available in the public domain and these data are the basis for many in silico models of mutagenicity (e.g. DEREK Nexus and Leadscope).

A

review of these data indicates that the perceived mutagenic potential of this class of compounds is based on a number of non-reproducible positive findings in the bacterial mutagenicity assay and positive bacterial mutagenicity data on a series of compounds where formation of reactive halodimethylsulfides (HDMS) in DMSO may have compromised the interpretation of the Ames data (HDMS are typically mutagenic). The only genuine mutagenic, genotoxic and carcinogenic compound within the 50+ acyl/sulfonyl halides described herein, was dimethylcarbamic chloride, which is appropriately considered to be a potential human carcinogen. Some in silico systems, such as Derek Nexus, contain rules which detail that the activity of this class should be considered a false positive flag for mutagenicity and ideally any in silico structure activity rules for mutagenicity in other systems should be likewise addressed. The data presented here supports the view that these alerts should currently be interpreted as a false positive flag for mutagenicity and the entire class viewed as low concern from a mutagenicity perspective. The formation of these reactive halodimethylsulfides is an example of the classical Pummerer rearrangement. The chemical reactivity of this class of compounds also supports the contention that they are of limited concern from a mutagenic and carcinogenic impurity risk perspective when used in the synthesis of drug products. They can be expected to rapidly purge from any reaction sequence with generic predicted purge factors in the range 1x103 - 3x105/stage and hence should be effectively eliminated at the stage of introduction. We would therefore recommend avoiding DMSO as solvent for mutagenicity tests with acyl/sulfonyl halides due to the potential for false positive results arising from DMSO reaction products, which are not relevant in aqueous, physiological conditions. Furthermore, as indicated by the Ames test data for mesyl chloride/2-fluorobenzoyl chloride, even non-DMSO organic solvents may not be appropriate for certain members of this class (acyl/sulfonyl halides), suggesting that they may not be amenable to adequate testing in the Ames assay.

Key words: Acyl/sulfonyl halides, DMSO, halodimethylsulfides, HDMS, chlorodimethylsulfide, CDMS, mutagenicity, reactive, purging, false positives, Pummerer rearrangement



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1. INTRODUCTION Acyl/sulfonyl halides (also known as carboxylic/sulfonic acid halides) are reactive derivatives of the corresponding carboxylic/sulfonic acids. Compounds representing these chemical classes are extremely reactive intermediates and are often used in the chemical synthesis of many different organic compounds, such as ketones (Friedel-Crafts acylation), aldehydes (Rosenmund reduction), esters, amides etc1. The common chemical structures are shown in Scheme 1. Scheme 1 Common chemical structure of (a) acyl halides and (b) sulfonyl halides.

However, due to their great chemical reactivity this class of compounds is efficiently purged from the majority, if not all, common synthetic pathways. Indeed, some members of this class react violently with water and most decompose readily in the presence of water with the formation of the corresponding acid and copious quantities of the hydrogen halide (see Scheme 2). Scheme 2 Hydrolysis of (i) acyl halides and (ii) sulfonyl halides with water a) Reaction of carboxylic acid halides with water O

O R

C

X

+

Carboxylic acid halides

H

O

H

Water

R

C

OH

+

HX Hydrogen halides

Carboxylic acids

b) Reaction of sulfonic acid halides with water O

O R

S

X

O

O

+

Sulfonic acid halides

H

O

H

Water

R

S

OH

Sulfonic acids


+

HX Hydrogen halides

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Teasdale et al.2 recently reported on risk assessment strategies for control of genotoxic impurities in new chemical entities. The authors exemplified their approach using a series of case studies, including two focused on thionyl chloride. In both case studies, the purging of thionyl chloride (one of the most reactive of the sulfonic acid halides) from the synthetic pathway was assessed. In the first case study, the theoretical purge factor was 9 x 1012, with the extremely high reactivity of thionyl chloride to high pH/aqueous conditions dominating the purging factor in the first three stages (four stages in total). In the second case study, the theoretical purge factor was 1 x 108 (again, four stages in total). Therefore, it would seem logical, that all members of this class of compounds would be equally effectively purged from synthetic routes. In this manuscript the theoretical purge factors for representative members of this class of compounds have been assessed. In parallel, there is considerable genetic toxicity data available in the literature for the acyl/sulfonyl chemical class (specifically in the bacterial reverse mutation assay3 or “Ames test”) and approximately just under one half of the representatives of this class have been reported to give positive results in the presence and absence of rodent S9 metabolic activation in the Ames test 4-14 (see Table 1).



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Table 1 Ames mutagenicity data for acyl/sulfonyl halides taken from the literature. Selection criteria was based on 1) clear definition of which test solvent was used in the Ames assay 2) the substances having no additional confounding (Q)SAR flags for potential mutagenicity Vehicle

Ames result

Referencea

53056-20-5

DMSOb

-

4

1-(2Ethylbutyl)cyclohexane carbonyl chloride 2,2-Dimethylpropanoyl chloride

211515-46-7

DMSO

+

4

3282-30-2

DMSO

+

4

2-Ethylhexanoyl chloride

760-67-8

DMSO

-

4

2-Methylpropanoyl chloride

79-30-1

DMSO

-

4

4-Chlorobenzoyl chloride

122-01-0

DMSO, DMKc

-

4, 5

Heptanoyl chloride

2528-61-2

DMSO

-

4

Neodecanoyl chloride

40292-82-8

DMSO

-

4

Pentanoyl chloride

638-29-9

DMSO

-

4

Propanoyl chloride

79-03-8

DMSO

-

4

2-Bromoisobutyryl bromide

20769-85-1

DMFd

+, -

4

1,1,2,2,3,3,4,4,4Nonafluorobutane-1sulfonyl fluoride 4Acetamidobenzenesulfonyl chloride 2-Chlorobenzoyl chloride

375-72-4

DMK

-

4

121-60-8

DMK

-

5

609-65-4

DMK

-

5

Hexadecanoyl chloride

112-67-4

DMK

-

4

Methanesulfonyl chloride

124-63-0

DMK, H2Oe, MeOHf

+

4

Octadecanoyl chloride

112-76-5

DMK

-

4

1Methylcyclohexanecarbonyl chloride Sulfuryl chloride

2890-61-1

DMEg

-

4

7791-25-5

DME

-

4

Pent-4-enoyl chloride

39716-58-0

H2O

-

4

Systematic name

CAS RN.

(2,4-Dichlorophenyl)acetyl chloride

Structure


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4-Methylbenzenesulfonyl chloride

98-59-9

DMSO

+

6

Octanoyl chloride

111-64-8

DMSO

-,+

4, 7

2-Fluorobenzoyl chloride

393-52-2

DMSO

+

8

Butanoyl chloride

141-75-3

DMSO

-,+

4, 7

Dimethylcarbamic chloride

79-44-7

DMSO

+

9

2,4-Dichlorobenzoyl chloride

89-75-8

DMK

-

10

Benzoyl chloride

98-88-4

DMSO, DMK

+, - h

10, 11

Phenylacetyl chloride

103-80-0

DMSO

+

10

3-Phenylpropionyl chloride

645-45-4

DMSO

+

10

Thionyl chloride

7719-09-7

DMSO

+

12

2-Propanesulfonyl chloride

10147-37-2

DMSO

+

13

Acetyl chloride

75-36-5

DMK

-

14

a. The following references appear in text and in Table 1: 4. European Chemicals Agency http://echa.europa.eu/ Accessed on 04th October 2013,5. Japan Chemical Industry Ecology- Toxicology and Information Center, (1996) Mutagenicity test data of existing chemical substances based on the toxicity investigation of the industrial safety and health law, 6 OECD SIDS datasheet http://www.inchem.org/documents/sids/sids/98599.pdf Accessed on Nov 2013-12-09, 7 Zeiger, E.; Anderson, B.; Haworth, S.; Lawlor, T.; Mortelmans K. Environ..Mol. Mutagen..1992, 19, Supp. 21, 2141, 8. Haworth, S.; Lawlor, T.; Mortelmans, K.; Speck, W.; Zeiger E. Environ. Mutagen.1983, 19, Supp. 1, 3 -142, 9. Dunkel, V.C.; Zeiger, E.; Brusick, D.; McCoy, E.; McGregor, D.; Mortelmans, K.; Rosenkranz, H.S.; and Simmon,V.F. Environ. Mol. Mutagen. 1984, 6,1-254, 10. Ohkubo, T.; Goto, S.; Endo, O.; Hayashi, T.; Watanabe, E.; Endo, H. Kankyo Kagaku, 1996, 6, 533-540, 11. U.S. Environmental Protection Agency December .Hazard Characterization Document screening-level hazard characterization sponsored chemical. 2012. Benzoyl Chloride, 12. Seifried, H. E.; Seifried, R. M.; Clarke, J. J.; Junghans, T. B.; San R. H. C. Chem. Res. Toxicol.2006, 19, 627-644, 13. Tsuchiya, Y.; Watanabe, E. M.F.; Watanabe M. Water Sci. Technol.1992, 25, 123-130, b. DMSO (Dimethylsulfoxide), c. DMK (Acetone), d. DMF (Dimethyl formamide), e. H2O (Water), f. MeOH (Methanol), g. DME (Ethylene glycol dimethyl ether), h. Compound that gave contradictory results based on choice of solvent.



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As the Ames test is recognized as the key assay for the detection of DNA reactive mutagens (i.e. presumptive carcinogens) it has become a pivotal assay in various regulatory testing strategies15-17. During this time the solvent dimethylsulfoxide (DMSO) has become the overwhelming choice for use in the Ames test due its effective solubilizing properties miscibility with water and molten agar

21, 22

18-20

its relatively low anti-bacterial effects and its

. Typically the use of DMSO as a vehicle in the Ames test

does not represent an issue, however in certain cases (such as the Ames testing of reactive intermediates) the recommendations from the OECD guideline 471 (i.e. the “solvent/vehicle should not be suspected of chemical reaction with the test substance” need careful consideration). For example acyl/sulfonyl halides are known to react with DMSO to form alkyl halides23-26 via the well established Pummerer rearrangement27. Hence it was noteworthy that the majority of the positive Ames findings reported in the literature for this chemical class were associated with the use of DMSO as the test solvent in the presence and absence of rodent S9 metabolic activation (Table 1). This becomes of increasing importance when one realises that the (Q)SAR predictions for this chemical class in in silico systems such as DEREK28 and Leadscope29 etc. are based on this positive carboxylic/sulfonic halide Ames data from the literature, which are used as training data sets for these prediction models. To investigate the influence of various solvents/vehicles (e.g. DMSO, acetone, ethanol, water, etc) on the potential mutagenicity of acyl/sulfonyl halides, internal and external Ames data on this chemical class were collated and re-examined, and where appropriate specific halo compounds were re-tested in the five strain Ames, or the screening Ames II assay, using alternative solvents to DMSO. This was vital in order to avoid the potential Ames positive Pummer rearrangement27 products formed from reaction of DMSO with these acylating agents. This publication demonstrates that not only does the chemical reactivity of this class of compounds ensure effective purging from typical reaction pathways, but that the majority of these compounds are not Ames positive when tested using non-DMSO solvents. These findings suggest that acyl/sulfonyl halides have a low probability of being potential impurities in drug products and should not be considered presumptive DNA reactive mutagens and hence they represent a low mutagenic and carcinogenic risk when used in the synthesis of drug products.

2. MATERIALS AND METHODS 2.1. Ames II assay Study Design The individual chemicals were tested under similar conditions. The individual tests follow in general the Ames II instruction manual by Xenometrix by Endotell GmbH30. The final concentration in the assay was 4.5% v/v. Revertant colonies are detected by using bromocresol purple as indicator dye (purple wells  no revertant colonies; yellow wells  revertant colonies). It has been established that the Ames II assay


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possesses a high predictivity for a positive mutagenic response in the standard Ames protocol

31-34

. Full

experimental conditions are provided in SUPPORTING INFORMATION. 2.2. Five strain plate incorporation Ames assay Study Design The five strain Ames plate incorporation test was performed with Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2uvrA (pKM101) or TA102 in the presence and absence of S9-mix (± S9) in line with the experimental design outlined in the OECD 471 guideline. 2.3. Carboxylic/sulfonic acid halides All compounds that were tested in this study are summarized in Table 2. Materials were obtained from Sigma, Aldrich, ABCR and Fluka. Stock solutions of each compound were prepared (immediately prior to use) for the Ames II and five strain assay. Given the potential for acyl/sulfonyl halides to react with water, anhydrous solvents were used at a final concentration of 4% v/v in the Ames assay. 2.4. HPLC Analyses Quantification of chlorodimethylsulfide (CDMS, CH3SCH2Cl) in DMSO solutions was performed with a Waters HPLC system using an Uptisphere 5μm C18-HDO stationary phase (150  4.6 mm, 5 µm) coupled to an UV-detector (λmax 300 nm). The mobile phase comprised of solvent A (acetonitrile: water, 10:90 v/v) with 1ml/L trifluoroacetic acid and solvent B (acetonitrile: water, 90:10 v/v) with 1ml/L trifluoroacetic acid using a linear gradient of 60-100% v/v solvent B in 20 minutes. 50 µg/ml of each carboxylic acid chloride was dissolved in DMSO and incubated at 37oC for 1 hour, and any residual CDMS was analyzed by HPLC.

3. DISCUSSION The basis of these studies was to explore the influence of the solvent/vehicle on the outcome of Ames tests reported in the literature for a series of acyl/sulfonyl halides (Table 1). The most obvious outcome of the studies was that the positive results from the Ames II and five strain Ames assays were influenced by the solvent/vehicle that was used. Fifteen of the eighteen acyl/sulfonyl halides reported to be positive in the Ames test where DMSO was used as a vehicle, were subsequently shown to be negative in the five strain Ames test, or Ames II assay, when non-DMSO solvents/vehicles were used (Table 2).



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Table 2: Bacterial reverse mutation assay data (default five strain plate incorporation data unless marked) for acyl/sulfonyl halides taken from Sanofi-Aventis and GlaxoSmithKline databases (includes reference to external positive Ames data where appropriate). Vehicle

Ames result

Referencea

69399-79-7

DMSOb

-

GSK

6-[(4-Fluorophenyl)oxy]-3pyridinecarbonyl chloride

862089-10-9

MeCNc

-

GSK

1-Propanesulphonyl chloride

10147-36-1

MeCN

-

GSK

1,3-Benzodioxole-5-sulphonyl chloride

115010-10-1

DMSO, MeCN

+ , -$

GSK

1-Chloro-2-methyl-1-oxo-2propanyl acetate

40635-66-3

MeCN

-

GSK

2-(2-Phenylhydrazinylidene)propanedioyl dichloride

19288-90-5

DMSO

-

GSK

2-Chloro-3-pyridinecarbonyl chloride

49609-84-9

MeCN

-

GSK

2,2,3,3-Tetramethylcycloprop anecarbonyl chloride

24303-61-5

DMSO , THFd

+ , -$

GSK

2,6-Difluorobenzenesulphonyl chloride

60230-36-6

MeCN

-

GSK

2,6-Difluorobenzoyl chloride

18063-02-0

THF

-

GSK

2,2-Dimethylpropanoyl chloride*

3282-30-2

DMSO , MeCN

+ , -$

4

2-Fluorobenzoyl chloride*

393-52-2

DMSO , MeCN , THF, DMKe, H2Of

+ , + , +, +, -

GSK , 8 , Sanofi,

2-Propanesulphonyl chloride*

10147-37-2

DMSO, MeCN

+ , -$

13

3-(2-Chlorophenyl)-5-methyl-4isoxazolecarbonyl chloride

25629-50-9

DMSO

-

GSK

3-Chloro-4-fluorobenzoyl chloride

65055-17-6

DMSO, MeCN

+ , -$

GSK

3-Phenylpropionyl chloride*

645-45-4

DMSO, MeCN

+ , -$

10

Systematic name

CAS RN.

3-(2-Chloro-6-fluorophenyl)-5methylisoxazole-4-carbonyl chloride

Structure


,GSK

, GSK

, GSK

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4-(1,1-Dimethylethyl) benzenesulphonyl chloride

15084-51-2

DMSO, MeCN

+ , -$

GSK

4-Acetylbenzenesulphonyl chloride

1788-10-9

MeCN

-

GSK

4-Fluorobenzenesulphonyl chloride

349-88-2

MeCN

-

GSK

5-Chloro-3-methyl-2-benzo[b] thiophenesulphonyl chloride

166964-33-6

DMSO, NMPg

+ , -$

GSK

5-Chloro-2-thiophenesuphonyl chloride

2766-74-7

MeCN

-

GSK

6-Chloro-3-pyridinecarbonyl chloride

58757-38-3

MeCN

-

GSK

α,α-Dimethyl-3,5bis(trifluoromethyl) benzeneacetyl chloride

289686-69-7

MeCN

-

GSK

Benzenesulphonyl chloride*

98-09-9

MeCN

-

GSK

Cyclopropanecarbonyl chloride

4023-34-1

MeCN

-

GSK

Dimethylcarbamic chloride*

79-44-7

DMSO , MeCN

+,+

GSK, 9

+, +, +, +, +,-

GSK, Sanofi, 4

+ , -$

GSK, 10

+ , -$

GSK, 12

MeCN , MeOHh , DMSO, DMK , H2O DMSO , MeCN DMSO , MeCN

Methanesulphonyl chloride*

124-63-0


103-80-0

Thionyl chloride*

7719-09-7

4-Methoxybenzoyl chloride*

100-07-2

MeCN

-

Sanofi

2-Methylbenzoyl chloride*

933-88-0

DMSO

-

Sanofi

2-(Butylsulfonylamino)benzoyl chloride

No-CAS

MeCN

-

Sanofi

3, 4-Dichlorobenzoyl chloride*

3024-72-4

DMSO

-

Sanofi

3-Chloro-4methylbenzenesulfonyl chloride*

42413-03-6

DMK

-

Sanofi

Acetyl chloride †

75-36-5

DMSO , H2O

+,-

Sanofi

Benzoyl chloride †

98-88-4

DMSO , H2O

+,-

Sanofi

Butanoyl chloride †

141-75-3

DMSO , H2O

+,-

Sanofi



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 25

4-Methylbenzenesulphonyl chloride †

98-59-9

DMSO , EtOHi

+,-

Sanofi

Octanoyl chloride †

111-64-8

DMSO , H2O

+,-

Sanofi

a.The following references appear in text and in Table 1: 4. European Chemicals Agency http://echa.europa.eu/. Accessed on 04th October 2013, 8. Haworth, S.; Lawlor, T.; Mortelmans, K.; Speck, W.; Zeiger E. Environ. Mutagen.1983, 19, Supp. 1, 3 -142 9. Dunkel, V.C.; Zeiger, E.; Brusick, D.; McCoy, E.; McGregor, D.; Mortelmans, K.; Rosenkranz, H.S.; Simmon,V.F. Environ. Mol. Mutagen. 1984, 6,1-254, 10. Ohkubo, T.; Goto, S.; Endo, O.; Hayashi, T.; Watanabe, E.; Endo, H. Kankyo Kagaku, 1996, 6, 533-540, 12. Seifried, H. E.; Seifried, R. M.; Clarke, J. J.; Junghans, T. B.; San R. H. C. Chem.Res. Toxicol.2006, 19, 627-644, 13. Tsuchiya, Y.; Watanabe, E. M.F.; Watanabe M. Water Sci. Technol.1992, 25, 123-130.b. DMSO (Dimethylsulfoxide), c. MeCN (Acetonitrile), d. THF (Tetrahydrofuran), e. DMK (Acetone), f. H2O (Water), g. NMP (N-Methylpyrrolidone), h. MeOH (Methanol), i. EtOH (Ethanol) *Compounds that were also tested in both the Ames II assay with positive results where DMSO was used as the test vehicle (demonstrating concordance with the standard 5 strain plate incorporation assay) and negative results when water or ethanol was used as the test vehicle, † Compounds that were tested in the Ames II assay only, $ Compounds that gave contradictory results based on choice of vehicle/solvent.


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We considered that the Ames positive results may be attributable to the Pummerer rearrangement27 product halomethylmethylsulfide (also known as halodimethylsulfide or HDMS) which presumably arises from the acylation of DMSO with an acyl or sulfonyl halides. Michelot et al.20 reported that acyl chlorides react with DMSO to yield the corresponding carboxylic acids and chlorodimethylsulfide (CDMS). Likewise, Boyle21 reported sulfonic acid chlorides can also react with DMSO to form CDMS as shown in scheme 3. Thea and Cevasco22 provided a much more detailed mechanistic understanding. They postulated an initial nucleophilic attack from the oxygen of the DMSO onto the acyl halide leading to acyloxysulfonium halide. Subsequently, hydrogen abstraction from one of the methyl groups occurs (either via a second DMSO molecule, an added base or X-) yielding an ylid. Removal of the carboxylate ion followed by addition of halide ion to the intermediate cation yields the halogenated dimethyl sulfide (HDMS). Bordwell and Pitt23 proposed a slightly different mechanism involving an extra step, whereby the halide ion (X-) displaces ArCO2- from the acyloxysulfonium halide yielding the corresponding sulfonium halide ion (MeS+(Cl)Me), which undergoes base (ArCO2-) catalysed dehydrochlorination to form the halogenated dimethyl sulfide (HDMS). The reaction of DMSO with sulfonyl halides to give the corresponding sulfonic acid and halogenated-dimethylsulfide (HDMS) is summarised within the general reaction mechanism below (Scheme 3).

Scheme 3: Pummerer Reaction exemplified using reaction between sulfonyl halides and DMSO



To confirm whether CDMS was formed with DMSO under the in vitro conditions prevailing for either versions of the Ames assay a series of acyl chlorides were dissolved in DMSO using standard assay conditions and incubated for 1 hour prior to HPLC determination for residual CDMS. The HPLC analysis demonstrated that even though the incubation time was greatly reduced compared to that used in the Ames assay itself, dissolving the acyl chlorides (acetyl-, octanoyl-, benzoyl-, phenylacetyl- and 3phenylpropionyl chloride) in DMSO at a concentration of 50µg/ml (i.e. the test concentrations used in the Ames II assay), produced CDMS in all samples. Initial concentrations formed within the first hour of incubation were in the range of 1.6 and 1.8 µg/ml (see Figure 1).

Figure 1 Analytical detection of chlorodimethylsulfide (CDMS) formation. Each acyl chloride (50 µg/mL) was dissolved in DMSO and incubated for 1 hour at 37oC then CDMS was analyzed by HPLC

2.0 1.78

1.84

1.77

1.72 1.62

Chlorodimethylsulfide-Conc. [µg/ml]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 25

1.5

1.0

0.5

0.0 Acetyl chloride

Octanoyl chloride

Benzoyl chloride


Phenylpropionyl chloride

From a chemical perspective, as CDMS is an alkylating agent it would be expected to produce a positive response in the Ames II/Ames test. The Ames II study confirmed that CDMS was positive in the TAMix strains when tested using DMSO as the vehicle (equivalent to TA100) (± S9) and TA98 strain (- S9). CDMS was also shown to be clearly positive in the standard Ames assay in TA100 and WP2uvrA strains (± S9) equivocal in the TA98 strain and negative in all of the other strains (see SUPPORTING INFORMATION). These studies on CDMS corroborated the published findings of Vito et al. 35 who reported it as positive in TA100 (± S9) and TA102 (+S9) and provided evidence that CDMS formation could have influenced previous Ames results for acyl/sulfonyl halides.


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The structural properties of the side chain (alkyl or aryl), molecular weight and chain length appeared to have no influence on the direct or indirect mutagenic potential (i.e. via formation of HDMS) within both chemical classes. In addition, structural environment, molecular weight, chain length and log P appear to have no influence within both chemical classes (data not shown). Therefore, one can conclude that the molecular environment adjacent to the carboxylic/sulfonic acid group has minimal influence on the formation of the mutagenic HDMS under the conditions of concentration and time used for preparation of the samples. The potential formation of HDMS in DMSO was the most significant difference correlating the Ames result with solvent used. The three exceptions to these general findings were 2-fluorobenzoyl chloride, methanesulfonyl chloride and dimethylcarbamic chloride which were all shown to be mutagenic when non-DMSO organic solvents were used. 2-fluorobenzoyl chloride was previously reported to be positive in the Ames when DMSO was used as the vehicle8 and in these current studies it was Ames positive when acetonitrile, tetrahydrofuran and acetone were used as alternative vehicles, but was clearly negative in the Ames II when water was used as a vehicle (which may be explained by its decomposition in water to the corresponding alcohol). Despite the fact that the structurally similar 2-chlorobenzoyl chloride was reported to be Ames negative when acetone was used as a vehicle5, 2-fluorobenzoyl chloride appears to be a genuine in vitro mutagen, of unknown in vivo genotoxic and carcinogenic potential. Methanesulfonyl chloride (MsCl) was reported to be positive in the Ames when acetone and methanol were used as the vehicle and in these studies it was positive in the Ames when DMSO, acetone and acetonitrile were used as solvents. When one considers that MsCl could be expected to react with each of these organic solvents to form either known or potentially DNA reactive intermediates (i.e. with acetone to form isopropenyl sulfonate, with methanol to form methylmethane sulfonate4, with DMSO to form CDMS, and with acetonitrile to form methyl sulfonyl nitrilium intermediates) then the Ames data becomes of questionable value in terms of assessing the compound‟s intrinsic mutagenicity. For instance, when methanol was inadvertently used as a vehicle during the mutagenicity testing of MsCl, the resulting data set was considered invalid within ECHA as the potential formation of the known mutagen methyl methane sulfonate would have compromised the overall test result4. However, although MsCl was clearly negative in the Ames II when water was used as a vehicle in these studies, the compound has also been reported to be weakly positive in the standard Ames assay when water was used as a vehicle4. The ambiguity surrounding the Ames data when water is used as a vehicle may be attributable to its „relative‟ stability in water. If the samples were prepared immediately prior to the test and run then there is a high likelihood that MsCl will remain intact; however, samples prepared some time prior to the Ames Test



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Page 16 of 25

may have hydrolysed to the corresponding, non-mutagenic alcohol. MsCl was therefore considered to be an equivocal (i.e. non-reproducible) Ames mutagen. Dimethylcarbamic chloride (DMCC) was reported to be positive in the Ames in Salmonella TA98, TA100, TA1535 and TA1537 strains when DMSO was used as a vehicle9 however it was also positive in the Salmonella TA98, TA100 strains when acetonitrile was used as vehicle in these studies. The positive findings using acetonitrile as vehicle, and the fact that CDMS itself is not mutagenic in Salmonella strains TA1535 and TA1537 support the position that DMCC is a genuine mutagen (perhaps acting via the carbamoyl moiety). While, DMCC was clearly negative in the Ames II in these studies when water was used as a vehicle (which would be expected given its rapid decomposition in water) given that the compound induces DNA adducts and micronuclei in vivo and is a documented rodent carcinogen36 the available data indicates that it is a genuine genotoxic carcinogen. Using the principles of ICH M737 the acceptable lifetime acceptable intake for DMCC would be approximately 0.6 µg/day (i.e. below the generic Threshold of Toxicological Concern of 1.5 µg/day) based upon the available carcinogenicity data from the carcinogenic potency database38 using the agreed formula outlined in ICH M7 (i.e. Lifetime AI = TD50/50,000 x 50kg). Acyl/sulfonyl halides are often used in chemical synthesis as reactive intermediates. An important aspect of these synthetic reactions is the use of anhydrous conditions, to minimise hydrolysis rates of these reactive intermediates, which can result in poor yields. However, whilst the hydrolysis rates of some aliphatic acyl chlorides are rapid39 (e.g. thionyl chloride reacts violently with water)40, it is known that several acyl/sulfonyl halides have rate constants that demonstrate that they are appreciably stable under aqueous conditions41. Many publications42,43 describe the reactions with water yielding the corresponding carboxylic/sulfonic acids and liberating the appropriate hydrogen halides (see Scheme 2). All of the end products of these hydrolysis reactions would not be predicted to be DNA reactive mutagens (i.e. carboxylic/sulfonic acids and hydrogen halides). The potential formation of CDMS/HDMS in DMSO was the most significant difference in the reaction of acyl/sulfonyl chlorides/halides with the solvents used in the Ames/Ames II test. Similar considerations are germane when considering alcoholic solvents, e.g. ethanol as the vehicle for Ames testing. Acyl/sulfonyl halides could react with alcoholic solvents to form the corresponding, nongenotoxic ester. However, most esterification reactions involving acyl/sulfonyl halides are typically base catalysed (removes evolved HCl from reaction mixture), e.g. pyridine, N-methylmorpholine, tertiary amines, quaternary ammonium salts44 and in the absence of a catalyst the reaction rate will be much slower. Bentley et al.45 evaluated the SN2 mechanism for alcoholysis, aminolysis and hydrolysis of acyl chlorides. They demonstrated that benzoyl chlorides (4-chlorobenzoyl chloride, 2-fluorobenzoyl chloride


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and benzoyl fluoride were used in the present investigations) were much more stable to alcoholysis than the corresponding alkyl chlorides. Finally, theoretical purge factor for all of the acyl/sulfonyl halides used in this study were determined using the method of Teasdale et al. 46 (Table 3). Table 3 Summary of Physicochemical Data for (a) acyl halides, (b) sulfonyl halides

CAS No

Boiling Point (°C)

Acetyl chloride

75-36-5

52

Acetyl bromide

506-96-7

75

Acetyl iodide

507-02-8

108

Dichloroacetyl chloride

79-36-7

106

Compounds

Reactivity

Solubility

Volatility

100

101

10

100

101

10

100

101

3

100

101

3

100

101

1

100

101

3

100

101

1

100

101

1

Cl

100

101

1

Br

100

101

1

100

101

1

100

101

10

100

101

10

100

101

3

Structure

A) Acyl halide O C

Cl

O C

Br

O C

I

O C

Cl

Cl

Cl

Dimethylcarbamoyl chloride

79-44-7

165

Butyryl chloride

141-75-3

102

O N

C

Cl

O C

Cl

O

Octanoyl chloride

111-64-8

195

Benzoyl fluoride

455-32-3

159

Benzoyl chloride

98-88-4

197

Benzoyl bromide

618-32-6

201

4-Chlorobenzoyl chloride

122-01-0

102

4-Fluorophenylacetyl chloride

459-04-1

4-(Trifluoromethoxy)benzoyl chloride

36823-888

55


103-80-0

94

C

Cl

O C

F

O C

O C

O C

Cl

Cl

F

60

O C

Cl O C

F F

F

Cl

O

O C

Cl



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O

3-Phenylpropionyl chloride

645-45-4

232

2-Fluorobenzoyl chloride

393-52-2

90

2,2,3,3-tetramethylCyclopropanecarbonyl chloride

24303-615

Page 18 of 25

100

101

1

Cl

100

101

10

Cl

100

101

10

100

101

10

100

101

1

100

101

10

100

101

3

100

101

1

10

101

1

100

101

1

100

101

10

100

101

10

100

101

1

100

101

1

100

101

1

C

Cl

O C F

2-(3,5-bis (trifluoro289686methyl)phenyl)-2-methyl 69-7 -propanoyl chloride

NA O

F

F

F

NA

O F F

Cl F O

2-Acetoxyisobutyroyl chloride

40635-663

6-(4-fluorophenoxy) nicotinic chloride

86208910-9

NA

Pivaloyl chloride

3282-30-2

105

Methanesulfonyl fluoride

558-25-8

123

Methanesulfonyl chloride

124-63-0

161

Methanesulfonyl bromide

41138-925

187

Iso propylsulfonyl chloride

10147-372

74

Trifluoromethanesulfonyl chloride

421-83-0

32

Benzenesulfonyl fluoride

368-43-4

207

Benzenesulfonyl chloride

98-09-9

177

O O

179

Cl

Cl F O

O

N

O Cl

(B) Sulfonyl Chlorides

4-Toluenesulfonyl chloride

O

O S

F

O

O S

Cl O

O S

Br O

O S

Cl

O

O S

F F

Cl

F

O

O S

O

O S

134

Cl

O

O

98-59-9

F

S

Cl


Page 19 of 25

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4-Chlorobenzenesulfonyl chloride

O

O

98-60-2

S

141

Cl

100

101

1

100

101

3

100

101

1

100

101

1

100

101a

10

100

101

10

100

101

1

100

101

1

Cl

Cl O

4-Fluorobenzenesulfonyl chloride

349-88-2

4-tert-Butylbenzenesulfonyl chloride

15084-512

S

95

O F

O

O S

165

Cl

O

O S

4-Acetylbenzenesulfonyl chloride

Cl

1788-10-9

339 O

O

Thionyl chloride

7719-09-7

79

1-Propanesulfonyl chloride

10147-361

78

1,3-Benzodioxole-5sulfonyl chloride

11501010-1

326

5-Chloro-3-methyl benzo[b]thiophene-2sulfonyl chloride

16696433-6

a

S Cl

Cl

O S O Cl

O Cl

O

S O

O

Cl

414

O S

S O

Cl

reacts violently in the presence of water

In all cases the analytes were deemed to be highly reactive (R=100) by design. Similarly, the solubility (S) was determined to be highly soluble (S=10), on the basis that in most cases the analytes spontaneously decompose in the presence of water, often violently (literature reports). While methanesulfonyl chloride is structurally similar to thionyl chloride and sulfuryl chloride the stability of these compounds is quite different; thionyl chloride and sulfuryl chloride are known to readily hydrolyze to SO2 and HCl whereas methanesulfonyl chloride hydrolysis rate is much slower47. As a result, the toxicological mechanism of action of these three compounds are considered to be different (i.e. driven by the hydrolysis products of thionyl chloride and sulfuryl chloride as opposed to the parent compound methanesulfonyl chloride itself). Interestingly, despite methanesulfonyl chloride‟s greater stability in water, the compound was still negative when the Ames test was conducted using water as the solvent (see Table 2). The volatility (V) was determined by comparing the literature boiling point (if one was available) of the acyl/sulfonyl halides with the mean boiling point of class 2 and 3 solvents (class 1 solvent were excluded on toxicological basis) recorded in ICH Q3C48. This was assessed as being 111°C (mean of 43 class 2/3



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Page 20 of 25

ICH Q3C solvents) and on that basis the acyl/sulfonyl halides were given either a high volatility (V=10. Boiling point > 20°C below that of a generic reaction/process solvent, with boiling point of 111°C), medium volatility (V=3. Boiling point ± 10°C that of a generic reaction/process solvent, with boiling point of 111°C), or low volatility (V=1. Boiling point > 20°C above that of a generic reaction/process solvent, with boiling point of 111°C). Finally, we were not able to generically assess either ionizability or use of physical processes (unique to the synthesis) and these factors were given a default value of 1. The data are summarised in Table 1. Of the 37 acyl/sulfonyl halides, 18 (49%) had a generic purging factor/stage of 1x103, 6 (16%) had a generic purging factor/stage of 3x104 and the remainder (33%) had a generic purging factor/stage of 1x105. The one exception was MsCl, which because of its greater hydrolytical stability will likely have a purging factor of 1x102/stage. This data strongly supports the contention that all acyl/sulfonyl halides would be purged within 2 stages (if not 1) of their introduction into the synthetic route. 4. CONCLUSION There is substantial mutagenicity data on acyl/sulfonyl halides available in well established online databases (e.g. CHEMID, NTP, ECHA etc) and in the literature. These data are the basis for many in silico models of mutagenicity (e.g. DEREK and Leadscope) which form the first step in any in silico assessment of potential impurities in a drug substance. A review of this data indicates that the perceived mutagenic potential of this class of compounds is based on a number of non-reproducible positive findings in the bacterial mutagenicity assay (Table 1) and positive bacterial mutagenicity data on a series of compounds where formation of reactive HDMS in DMSO via the Pummerer rearrangement may have compromised the interpretation of the Ames data (Tables 1 and 2). The only genuine mutagenic, genotoxic and carcinogenic compound within the 50+ acyl/sulfonyl halides described herein, was dimethylcarbamic chloride, which is appropriately considered to be a potential human carcinogen. Some in silico systems, such as Derek Nexus, contain rules which detail that the activity of this class should be considered a false positive flag for mutagenicity and ideally any in silico SAR rules for mutagenicity in other systems should be likewise addressed. The data presented here supports the view that these alerts should currently be interpreted as a false positive flag for mutagenicity and hence acyl/sulfonyl halides viewed as low concern from a mutagenicity perspective. Interestingly Snodin 48 recently discussed similar solvent issues with a different chemical class and indicated use of non-hydroxylic, non-reactive solvents such as hexane may be more appropriate to assess the intrinsic mutagenicity of alkyl chlorides. The in vitro situation could be even more complicated than discussed. In the presence of an alcoholic vehicle, the evolved HCl generated as a by-product (since no base is present to neutralize the acid formed) could further react with the corresponding alcohol to form a chloroalkane (e.g. chloromethane, chloroethane and


Page 21 of 25

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2-chloropropane) all of which are known mutagens that again could compromise the overall mutagenicity test results of the parent acid chloride being assessed. The chemical reactivity of this class of compounds also supports the contention that they are of limited concern from a genotoxic risk perspective. They can be expected to rapidly purge from any reaction sequence with generic predicted purge factors in the range 1 x 103-3x105/stage and hence are candidates for control under ICH M7 option 4 as they should be effectively eliminated at the stage of introduction. We would therefore recommend avoiding DMSO as solvent for mutagenicity tests with acyl/sulfonyl halides due to the potential for false positive results arising from DMSO reaction products, which are not relevant in aqueous, physiological conditions. Furthermore, as indicated by the Ames test data for MsCl/2-fluorobenzoyl chloride, even non-DMSO organic solvents may not be appropriate for certain members of this class, suggesting that they may not be amenable to adequate testing in the Ames assay. Careful consideration of an appropriate solvent for a test material is clearly aligned with the Bacterial Reverse Mutation Test OECD guidance 471 and it should be an important factor when conducting Ames tests on reactive chemical intermediates in relation to the control of mutagenic impurities in drug products. It would be extremely useful to „map out‟ the chemical space of these very reactive exceptions e.g., MsCl/2-fluorobenzoyl chloride to this general rule (that testing of this class of compounds using nonDMSO organic solvents will produce negative Ames test outcomes). In particular, the discussion could be broadened to some extent by assessing other highly reactive reagents such as pyrophosphoryl chloride, phosphoryl chloride (POCl3) and phosphorus trichloride (PCl3) that are reported to be Ames negative 51

4, 50,

. We would therefore request interested parties to lodge relevant Ames test data in DMSO, non-DMSO

organic solvents and water, with Lhasa Ltd., as part of the Derek nexus consortium, to help improve the (Q)SAR mutagenicity predictions for these chemically reactive compounds.

ACKNOWLEDGEMENTS The authors would like to acknowledge the extremely useful comments made by both the reviewers and editor-in-chief during the review phase of the manuscript. In addition we would like to acknowledge the help and support of Dr Mike Urquhart who provided extremely useful commentary on the Pummerer rearrangement.



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Page 22 of 25

SUPPORTING INFORMATION Detailed individual revertant colony counts for the GSK bacterial reverse mutation (Ames) plate incorporation studies referenced in Table 2 and Figure 1 are available. Details of positive and negative controls and subsequent fold changes are also presented. All studies were conducted in accordance with OECD 471 guidelines. This information is available free of charge via the Internet at http://pubs.acs.org/.

REFERENCES 1.Sonntag, N.O.V. Chem. Rev., 1953, 52, 237-416. 2. Teasdale, A.; Elder, D.; Chang, S-J.; Wang, S.; Thompson, R.; Benz, N.; Sanchez Flores, I.H., Org. Process Res. Dev. 2013, 17, 221-230. 3. OECD Guideline For Testing Of Chemicals 471 Bacterial Reverse Mutation Test Adopted: 21st July, 1997 1/11. 4. European Chemicals Agency http://echa.europa.eu/ Accessed on 04th October 2013. 5. Japan Chemical Industry Ecology- Toxicology and Information Center, (1996) Mutagenicity test data of existing chemical substances based on the toxicity investigation of the industrial safety and health law. 6. OECD SIDS datasheet. http://www.inchem.org/documents/sids/sids/98599.pdf Accessed on Nov 2013. 7. Zeiger, E.; Anderson, B.; Haworth, S.; Lawlor, T.; Mortelmans K. Environ. Mol.Mutagen. 1992, 19, Supp. 21, 2-141. 8. Haworth, S.; Lawlor, T.; Mortelmans, K.; Speck, W.; Zeiger E. Environ. Mutagen.1983, 19, Supp. 1, 3 -142. 9. Dunkel, V.C.; Zeiger, E.; Brusick, D.; McCoy, E.; McGregor, D.; Mortelmans, K.; Rosenkranz, H.S.; Simmon,V.F. Environ. Mol. Mutagen. 1984, 6,1-254.


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10. Ohkubo, T.; Goto, S.; Endo, O.; Hayashi, T.; Watanabe, E.; Endo, H. Kankyo Kagaku, 1996, 6, 533540. 11. U.S. Environmental Protection Agency December .Hazard Characterization Document screeninglevel hazard characterization sponsored chemical. 2012. Benzoyl Chloride. 12. Seifried, H. E.; Seifried, R. M.; Clarke, J. J.; Junghans, T. B.; San R. H. C. Chem. Res. Toxicol.2006, 19, 627-644. 13. Tsuchiya, Y.; Watanabe, E. M.F.; Watanabe M. Water Sci. Technol.1992, 25, 123-130. 14. Zeiger, E.; Anderson, B.; Haworth, S.; Lawlor, T.; Mortelmans, K. Environ. Mol.Mutagen.1998, Supp. 11, 12, 1-158. 15. Riccio E.S.; Mortelmans K. Mutat. Res., 2000, 455, 61–69. 16. Ames, B.N.; McCann, J.; Yamasaki E. Mutat. Res., 1975, 31, 217–233. 17. Mortelmans, K.; Zeiger E. Mutat. Res., 2000, 455, 29–60. 18. Lipinski, C.A. J. Pharmacol. Toxicol. Methods 2000, 44, 235-249. 19. Alsenz, J.; Kansy, M. Adv. Drug Delivery Rev. 2007, 59, 546-567. 20. Balain, K.V.; Savchuck, N.P.; Tetko, I.V. Curr. Med. Chem., 2006, 13, 223-241. 21. Gatehouse, D.; Haworth, S.; Cebula, T.; Gocke, E.; Kier, L.; Matsushima, T.; Melcion, C.; Nohmi, T.; Ohta, T.; Vennit, S.; Zieger, E. Mutat. Res., 1994, 312, 217-233. 22. Maron, D.M.; Katzenellenbogen, J.; Ames B.N. Mutat. Res., 1981, 88, 343-350. 23. Michelot R.; Tchoubar B. Bull. Soc. Chem. France, 1966, 3039-3040. 24. Boyle R.E. J. Org. Chem., 1966, 31, 3880–3882. 25. Thea, S.; Cevasco, G. J. Org. Chem., 1988, 53, 4121-4122. 26. Bordwell, F.G.; Pitt, B.M. J. Am. Chem. Soc. 1955, 77, 572.



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27. March,J. Ed. Advanced Organic Chemistry, 4th Edition, Wiley Intersience, New York, 1992, pp. 1236. 28. DEREK, Lhasa Ltd., 2013. https://www.lhasalimited.org/derek_nexus/. Accessed on 05th November 2013. 29. Leadscope, 2013. http://www.leadscope.com/. Accessed on 05th November 2013. 30. http://www.xenometrix.ch/. Accessed on 05th November 2013. 31. Flückiger-Isler, S.; Baumeister, M.; Braun, K.; Gervais, V.; Hasler-Nguyen, N.; Reimann, R.; Van Gompel, J.; Wunderlich, H.G.; Engelhardt, G. Mutat. Res., 2004, 558, 181-197. 32. Kamber, M.; Flückiger-Isler, S.; Engelhardt, G.; Jaeckh, R.; Zeiger E. Mutagenesis, 2009, 24, 359366. 33. Gee, P.; Maron, D.M.; Ames B.N. Proc. Natl. Acad. Sci. U.S.A., 1994, 91, 11606–11610. 34. Gee, P.; Sommers, C.H.;Melick, A.S.; Gidrol, X.M.; Todd, M.D.; Burris, R.B.; Nelson, M.E.; Klemm, R.C.; Zeiger E. Mutat. Res., 1998, 412, 115 – 130. 35. Vito, M.; Esposito, G.; Vicari, L.; Lembo S.; Imperatrice, M.L.; De Marinis E. Boll. Soc. Ital. Sper. 1985, 61, 917-923. 36. International Agency for Research on Cancer Monograph, 2011. http://monographs.iarc.fr/ENG/Monographs/vol71/mono71-22.pdf. Accessed on 26th May 2015. 37. ICH M7, Step 4, 23rd June 2014. http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Multidisciplinary/M7/M7_Step _4.pdf. Accessed on 14th May 2015. 38. The Carcinogenicity Potency Database http://toxnet.nlm.nih.gov/cpdb/chempages/DIMETHYLCARBAMYL%20CHLORIDE.html. Accessed on 26th May 2015.


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