Facile synthesis of oligo anilines as permanent hair dyes: How chemical modifications impart colour and avoid toxicity

Article abstract of DOI:10.1039/c9nj03362a

Many dyes for long-lasting hair coloring contain aromatic amines that undergo oxidative polymerizations, resulting in allergic contact dermatitis, with the potential to develop serious toxic effects. Among these amines, para-phenylenediamine (PPD) is a small molecule form of aniline commonly used in beauty products despite being a known allergen to humans. Hence, in this study we designed and synthesized safer PPD analogues through the synthesis of oligomeric PPD and the introduction of bulky side chains on PPD to overcome the PPD dye's toxicity. We hypothesized that (a) an increase in the molecular size of PPD by addition of the PPD monomer unit on free amines and (b) strategic functionalizations at the ortho position of PPD with strong electron-donating bulky groups are able to maintain the hair coloring properties, and increase the resistance to binding to skin proteins and therefore decrease the chance of skin sensitization. 13 oligomers were synthesized, with the aim to produce safer hair dyes while minimising eventual toxicity to humans. In particular, oligomers with bulky sidechains, PPD 6 (13), PPD 7 (14) and PPD 8 (15), displayed weak-to-moderate (27.1%, 24.1% and 34.0%) sensitization potential. The results confirmed the importance of having bulky and strong electron donating O-alkyl substituents in order to decrease the reactivity of PPD analogues towards skin proteins, thus preventing the interaction with immune cells and providing safer hair dyes.

Full text of DOI:10.1039/c9nj03362a

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DOI: 10.1039/C9NJ03362A
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Facile synthesis of oligo anilines as permanent hair dyes: How
chemical modifications impart colour and avoid toxicity
Received 00th January 20xx,
Accepted 00th January 20xx
Gopalakrishnan Venkatesana, Yuri Dancikb,c, Arup Sinhad, Mei Bigliardi b,e, Ramasamy Srinivasb,
Thomas Dawsonb, Suresh Valivaveettil*d, Paul Bigliardi*b,e, and Giorgia Pastorin*a
DOI: 10.1039/x0xx00000x
Many dyes for long-lasting hair coloring contain aromatic amines that undergo oxidative polymerizations, resulting in
allergic contact dermatitis, with potential to develop serious toxic effects. Among these amines, para-phenylenediamine
(PPD) is a small molecule form of aniline commonly used in beauty products despite being a known allergen to humans.
Hence, in this study we designed and synthesized safer PPD analogues through the synthesis of oligomeric PPD and the
introduction of bulky side chains on PPD to overcome the PPD dye’s toxicity. We hypothesized that a) increase in
molecular size of PPD by addition of the PPD monomer unit on free amines and b) strategic functionalizations at the ortho
position of PPD with strong electron-donating bulky groups are able to maintain the hair coloring properties, increase the
resistance to binding to skin proteins and therefore decrease the chance of skin sensitization. 13 oligomers were
synthesized, with the aim to produce safer hair dyes while minimising eventual toxicity to humans. In particular, oligomers
with bulky sidechain, PPD 6 (13), PPD 7 (14) & PPD 8 (15) displayed a weak-to-moderate (27.1%, 24.1% & 34.0%)
sensitization potential. The results confirmed the importance of having bulky and strong electron donating O-alkyl
substituents in order to decrease the reactivity of PPD analogues towards skin proteins and thus preventing the interaction
www.rsc.org/
with
immune
cells
and
providing
safer
hair
dyes.
application of PPD-containing hair dyes.6,7,8 Systemic PPD Hair dye
poisoning
Introduction
Many dyes for long-lasting and intense hair coloring contain
unstable di–/tri-functional aromatic amines that have to undergo
oxidative polymerizations to provide the desired pigmentation.1
However, oxidative and enzymatic alterations of these amines
generate potentially harmful and sensitizing metabolites which may
cause serious irritant and allergic contact dermatitis.2,3 Para-
phenylenediamine (PPD) (Figure 1) is a common amine-containing
compound used in beauty products.4 In the year 2006 it was
declared as “allergen of the year”.5 This small molecule (MW =
108.1 Da) is able to penetrate skin and, in clinical studies, has been
detected in plasma, urine and faeces of volunteers upon topical
NH2
NH2
H
R
N
R1HN
NHR2
NH2
NH2
Scaffold I
R1, R2 = -H, R1, R2 = -CH2-Ar-R3
R3 = -4-OH, 4-NO2, 4-OCH3, 4-CH3
2-(Methoxymethyl)benzene-
1,4-diamine, (ME-PPD), R= -CH2-O-CH3
2,5-Diaminotoluene (PTD), R= -CH3
H
p-Phenylenediamine
NHR1
OR2
H
N
H
N
N
NH2
R1
H2N
N
H
H2N
N
H
n=0,1
n=1,2
NH2
Scaffold II
R1= -H, R2 = -CH2(CH2)4CH3,
Scaffold IV
R1 = -CH2-Ar-CH3
Scaffold III
-CH2CH(Et)(CH2)3CH3
R1, R2 = -CH2(CH2)4CH3
Figure 1. Structures of PPD derivatives.
a. Department of Pharmacy, National University of Singapore, Lower Kent Ridge
Road, 18 Science Drive 2, Singapore 117543
incidents such as cervico-facial oedema, severe rhabdomyolysis,
acute tubular necrosis and renal failure have also been reported.9
Till now, it has not been clearly established yet if PPD itself or its
oxidized metabolites, which include a spectrum of known and
unknown electrophiles, are responsible for the toxic, irritant or
allergic reactions and how these compounds affect the
keratinocytes in the skin and the immune response.10
Recently, hair dye products containing structurally-similar PPD
alternatives, such as para-toluenediamine sulfate (PTD) and 2-
(methoxymethyl)benzene-1,4-diamine (ME-PPD), have been
b. Skin Research Institute of Singapore, 8A Biomedical Grove, #06-06, Immunos,
Singapore 138648
c. Le Studium Loire Valley Institute of Advanced Studies, Orléans, France and
Université François-Rabelais de Tours, Faculté de Pharmacie, EA 6295
Nanomédicaments et Nanosondes, 31 avenue Monge, 37200 Tours, France.
d. Department of Chemistry, National University of Singapore, Lower Kent Ridge
Road, 18 Science Drive 2, Singapore 117543
e Department of Dermatology, Division of Dermato-Allergy, University of
Minnesota, 420 Delaware St SE, Minneapolis, MN 55455, USA.
* Corresponding authors. Contact email: phapg@nus.edu.sg; pbigliar@umn.edu
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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proposed as safer alternatives to PPD (Figure 1). While proteins. This idea was taken from studies in which thVeieiwntArroticdleuOctniloinne
DOI: 10.1039/C9NJ03362A
approximately half of PPD-sensitive individuals still reacted to of one or two electron-withdrawing chains at the ortho position to
PTD,11 ME-PPD showed lower sensitization potential than the main reactive functional group in a molecule (e.g. the ‘OH’
PPD.12 Nevertheless, small cohort studies still yielded a group in acetaminophen) resulted in a decreased analgesic effect
sensitizing response to ME-PPD in more than 30% of PPD- due to enhanced oxidation of the drug.17-18
Hence, by introducing
allergic subjects.12 Hence, more research and development to
functionalized sidechains or substituents at the ortho position of
achieve less irritant and sensitizing stable hair dyes are
warranted.
the PPD, our PPD analogues could decrease the benzene’s reactivity
towards oxidation and overcome the toxicity of PPD.
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Polyanilines are electro-conducting polymers and are the
model compounds to prepare oligoanilines. They have been
synthesized as nanofibers, nano-ribbons polymer materials for
detection of electro-conductance and for improved cell-
biomaterial interaction in tissue engineering applications.13-15
To our knowledge, polyanilines so far have been tested for
their material properties only and not as hair dyes.
In this study we designed and synthesized a series of
oligomeric structures of PPD by adding monomeric units of
PPD and introducing functionalized side chains or substituents
in order to increase the molecular size, lipophilicity and
hydrogen bonding properties of the hair dyeing molecule. We
further postulated that these structural modifications would
render the PPD derivatives more resistant to oxidative changes
compared to PPD, thereby decreasing their ability to interact
with skin proteins and induce skin sensitization. Figure 1
Our results confirm the hypothesis that a suitable combination of
increased molecular size and strong electron-donating side chains
minimize the skin sensitization potential and preserve the desired
hair nuance properties, making these oligomeric series of PPD
analogues potentially much safer in comparison to existing products
in the market.
Result and Discussion
Synthesis of PPD derivatives
All compounds were successfully synthesized, with yields in the 50-
1
99% range, and characterized by H and 13C NMR spectroscopy and
High Resolution Mass Spectroscopy (HRMS) (Figures S1-S14). The
PPD precursor dinitro derivative was obtained by the coupling of p-
nitroaniline with nitrobenzene in good yield (refer to experimental
section of SI for details). The secondary amine proton in 4,4′-
dinitrodiphenylamine resonates at
δ 9.98 ppm, showing a
illustrates the modifications made to PPD.
We increased the
significant upfield shift (Δδ 3.22 ppm) of the secondary amine
1
number of 1° and 2° amine groups by a) introducing novel
head groups and the conjugate system into a dimeric, trimeric
or tetrameric structure; b) adding long hydrocarbon chains c)
increasing the sum of hydrogen bond accepting and hydrogen
bong donating groups and d) increasing the molecular weight
proton in the H NMR spectrum of 4,4′-diaminodiphenylamine (1).
This upfield shift of the NH proton is due to the increase of the
electron density in the aromatic ring upon the conversion of the
nitro group into the amine. This is further supported by the upfield
shift of aromatic protons from the ranges δ 8.2-7.3 to δ 6.6-6.4.
The dimeric compound 1 (PPD 1) was synthesized by the reduction
of 4,4′-dinitrodiphenylamine with tin powder in strongly acidic
medium (Scheme 1).
.
Modifications specifically included
of the compounds
symmetrically amine (NH2/NH2) capped and aryl (Ph/NH2)
capped oligoanilines (Scaffold I), C-substituted and C,N-
substituted PPD derivatives (Scaffold II), symmetrically amine
(NH2/NH2) capped oligoanilines (Scaffold III), unsymmetrically
aryl(Ph/NH2) capped oligoanilines (Scaffold IV).
NHR'
H
N
H
N
R
Sn, Conc HCl
reflux, 18 h
Ar-CHO, p-TsOH, EtOH
r.t, over night
H2N
NH2
Ar
N
N
Ar
2-5
1 (78%)
NO2
R= -H, R'= -C6H5NO2
H
N
The design of dimer and trimer structures (Figure 1, Scaffolds I, III
and IV) was aimed at assessing how the increase in size and
lipophilicity affected the properties of the compounds, especially
when compared with the main product of oxidation of PPD,
Bandrowski’s base (BB). For the C-substituted and C,N-substituted
derivatives (Scaffold II), monomeric or dimeric PPD was substituted
with hexyl chain to investigate the effect of electron donating chain
NaBH4, THF/MeOH
r.t., 24 h
Ar
N
N
H
Ar
6-9
H
Entry Reagent (ArCHO)
Ar
Yield
1
2
3
4
4-hydroxy benzaldehyde
4-nitro benzaldehyde
4-methoxy benzaldehyde
4-methyl benzaldehyde
C6H4(4-OH)
67% (6)
82% (7)
88% (8)
90% (9)
C6H4 (4-NO2)
C6H4(4-OCH3)
C6H4 (4-CH3)
The long chain compounds were designed to investigate
on PPD.
the effect of the substitution at the ortho position of PPD: as it was
reported that the development of skin allergy decreases in the
order PPD < PTD < ME-PPD, 16 we reasoned that electron-donating
alkyl functional chains at the ortho position of PPD would decrease
the oxidation potential of the benzene ring in PPD, thereby
decreasing its conversion into a reactive quinonediamine; at the
same time, by increasing the nucleophilic property of the benzene
ring through electron donating resonance effects, we would have
further decreased PPD’s reactivity towards nucleophilic skin
Scheme 1. Synthesis of symmetrically amine (NH2/NH2) capped PPD
1(1), aryl (Ph/NH2) capped oligoanilines PPD 2-5 (6-9).
A host of higher oligomers (MW > 400 Da) of symmetric aryl capped
derivatives (PPD 2-5 (6-9)) was synthesized by functionalizing the
synthesized PPD 1. First, an imine (2-5) was synthesized by the
reaction of the symmetric PPD 1 with an aldehyde in presence of p-
toluenesulphonic acid.
2 | J. Name., 2012, 00, 1-3
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N1,N4-bis(4-nitrophenyl)-1,4-benzenediamine (18), VfieowlloArwticeledOnlibnye
DOI: 10.1039/C9NJ03362A
Then, the imine was reduced to provide the corresponding amines Sn/HCl reduction. Similarly, the reaction of compound 1 with two
(6-9) (Scheme 1) in very good yield (67-90%). Irrespective of the equivalents of 1-fluoro-4-nitrobenzene and subsequent reduction
nature of substituents, the imine proton signals appeared in the of resulting nitro compound 19 afforded 4'-bis[(4 aminophenyl)
range of δ 8.9 to 8.50 ppm, with the nitro derivative showing the amino] diphenylamine (PPD 11 (21)) (Scheme 3).
highest chemical shift. Upon reduction, the imine signal
5
6
7
8
NH2
n=1,2
NO2
H
9
disappeared and two new signals, corresponding to the NH–CH2
group, appeared in the range δ 5.9 – 5.4 ppm (triplet) for the amine
H
C6H4(CH3)CHO,
NH2
p-TsOH, EtOH
N
N
N
Et3N, DMSO
+
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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90oC,
r.t., over night
O2
N
N
O2
N
N
H
(n= 0)
H
n= 0,1
n=0,1
3 days, N2
NH
F
24
25
(n= 0)
(n= 1)
22
23
H
and δ 4.3
– 4.0 ppm (doublet) for the methylene group,
(n= 1)
H
H
H
H
N
N
N
N
respectively. As usual, all aromatic proton signals underwent an
upfield shift upon imine reduction.
Sn, Conc. HCl
reflux, 18 h
NaBH4, THF/MeOH
r.t., 24 h
H2
N
N
O2
N
N
H
H
n= 0,1
n= 0,1
28, (n= 0, 88%)
29, 72%
26
27
(n= 0)
(n= 1)
O2
N
F
NH2
NH2
NH2
NO2
(n= 1,
)
OR
OR
O2
N
NH2
OR
OH
SnCl2, Conc. HCl, EtOH
reflux, 24 h
Et3N, DMSO
90oC, 24 h
DMF, reflux, 18 h
N
H
NO2
NH2
Scheme 4. Synthesis of unsymmetrically aryl(Ph/NH2) capped
oligoanilines PPD 12(28) and 13(29)).
14
10-11
12
84 %
)
, (R= -CH2-(CH2)4-CH3,
13
CH3,
, (R= -CH2-CH(Et)-(CH2
) -
3
SnCl2, Conc.
HCl, EtOH,
reflux, 24 h
82%
)
DMF, reflux, 18 h
NHR1
OR
NHR1
OR
H2
N
NH2
OR
SnCl2, Conc. HCl, EtOH
reflux, 24 h
A different synthetic approach was adopted for the synthesis of the
unsymmetrically aryl capped oligoanilines (Scheme 4). Non-
symmetric intermediate oligomers (22-23) were synthesized with
amino and nitro groups as terminal substituents. The free amino
terminal group was substituted with para-tolyl methyl substituent
and the nitro terminal group was reduced to obtain non-symmetric
oligomers 28 and 29 (PPD 12 and 13). In our initial effort, we
synthesized a dimeric compound 4-amino-4′-nitrodiphenylamine
(22) by the coupling of PPD with one equivalent of 1-fluoro-4-
nitrobenzene in the presence of triethylamine. The secondary
amine signal appeared at δ 8.88 ppm. The corresponding imine (24)
was then synthesized by the reaction with para-
toluenecarboxaldehyde in the presence of para-toluenesulphonic
acid. Sodium borohydride reduction of the imine, followed by
Sn/HCl reduction of the nitro group, afforded non-symmetrically
N
H
NO2
NH2
, (R, R1= -CH2-(CH2
15
,
17
99%
)
)4-CH3,
16
85%
)
(R= -CH2-(CH2
)4-CH3,
Scheme 2. Synthesis of C-substituted derivatives PPD 6 (12), 7 (13)
and 9 (17) and C,N-substituted PPD 8 (15).
Alkoxy derivatives of PPD such as PPD 6-7 (12-13), 8(15) and PPD
9(17) (12-13, 15 and 17) were synthesized by nucleophilic
substitution of the bromo group of 1-bromoalkane with the in situ
generation of phenoxy derivative of 2-amino-5-nitrophenol.
Subsequent SnCl2 reduction of the nitro group afforded the 2-alkoxy
PPD 6 (12) in high yield (Scheme 2). Although O-alkylation was the
major product (12), for the 1-bromohexane N,O-alkylation was also
observed. N,O-hexyl-4-aminobenzene (17) was isolated as minor
product along with the O-alkylated product. Further, the newly
synthesized product 12 was further functionalized to get higher
oligomers. The substituted PPD derivative (12) was treated with 1-
fluoro-4-nitrobenzene in presence of triethylamine to get the nitro
derivatives, which were then reduced by SnCl2/Conc HCl to get the
corresponding dimer with O-hexyl substituted PPD 8 (15). In the
substituted
PPD
dimer
4-amino-4′-[(4-
tolylaminomethyl)phenyl]diphenylamine (PPD 12 (28)). The imine
proton signal of compound 24 appeared at δ 9.37 ppm, and then
disappeared upon sodium borohydride reduction. A triplet and a
doublet appeared at δ 6.24 and 4.22 ppm corresponding to the
amine and methylene proton, respectively, of the newly formed
NH–CH2 group in compound 26. Simultaneously, compound 28 was
coupled with 1-fluoro-4-nitrobenzene to get the nitro compound
N1-[4-(4-tolylaminomethyl)phenyl]-N4-(4-aminophenyl)-1,4-
O,N alkyl functional series, PPD
quantitative yield by reacting 2-amino-5-nitrophenol with
equivalents of 1-bromohexane, and subsequent reduction of nitro
compound.
9
(17) was obtained in
a
2
benzenediamine (27) with 26% yield, which was then reduced with
SnCl2/HCl
to
get
corresponding
trimer
N1-[4-(4-
tolylaminomethyl)phenyl]-N4-(4-aminophenyl)-1,4-benzenediamine
(PPD 13 (29)).
NH2
n=1,2
+ 2
NO2
H
H
N
NO2
N
NH2
Et3N, DMSO
Sn, Conc. HCl
90oC, 3 days, N2
O2
N
N
H
(n= 0)
(n= 1)
reflux, 18 h H2
N
N
H
n=1,2
NH
F
n=1,2
H
18,
19,
An alternative method was also developed for the synthesis of the
trimeric PPD 13 (29) with higher yield and involving less number of
steps. Similar to the synthesis of compound 22, non-symmetrically
20,
61%
50%
(n= 0,
(n= 1,
)
)
21,
Scheme 3. Synthesis of symmetrically amine (NH2/NH2) capped
oligoanilines PPD 10(20) and 11(21).
substituted
trimeric
structure
N1-(4-aminophenyl)-N4-(4-
nitrophenyl)-1,4-benzenediamine (23) was synthesized from the
NH2/NH2 type dimeric compound 1.
The higher oligomers (PPD 10 and 11 (20-21)) were conveniently
synthesized via SNAr type substitution reactions with aromatic
amines.19 PPD 10 (20) was synthesized by coupling PPD with two
equivalents of 1-fluoro-4-nitrobenzene to the dinitro compound
Compound
23
was
then
condensed
with
para-
toluenecarboxaldehyde to get the corresponding imine (25).
Sequential reduction of the imine by sodium borohydride yielded
compound 27 with high yield (95%) and the nitro groups of 27
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afforded PPD 13 (29) upon reduction with Tin powder in acidic density at ortho and para positions to O-hexyl chain (such as
View Article Online
DOI: 10.1039/C9NJ03362A
medium. A general synthetic pathway is presented in Scheme 4.
positions 3, 5 and 6 in PPD 6
and 7) in pi system of benzene ring and make it a more nucleophilic
Direct peptide reactivity assay (DPRA) for in chemico skin site than PTD and ME-PPD. This causes less interaction of our PPD
sensitization
derivatives with nucleophilic protein or peptides, as confirmed by
the DPRA.
9
In addition, we aimed to decrease the oxidation potential by
10
11
12
13
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16
17
18
19
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21
22
23
24
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27
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Table 1 summarizes our classification results for PPD, ME-PPD, PTD affecting the resonance mechanism on benzene ring and amine
and our PPD derivatives, following the Organisation for Economic chain, as previously reported.16-18 This was achieved by blocking the
Development’s (OECD) guidelines.20
extension of conjugation with either substituted benzyl chain (as in
S.
N
1
2
3
4
5
6
7
8
9
8
9
Compound
Cysteine % (1:10)
Lysine
(1:50)
33.2
23.7
15.6
11.3
8.5
12.8
13.4
15.4
14.3
10.8
9.0
%
Mean Peptide depletion % Sensitizing potential
Cysteine (1:10) / (Lysine1:50)
PPD
77.7
69.4
51.2
48.0
47.1
40.9
40.8
32.7
53.8
37.1
64.1
78.8
27.6
35.2
70.9
62.9
71.3
55.5
46.6
33.4
29.7
27.8
26.9
27.1
24.1
34.0
23.9
36.5
44.5
17.9
23.0
38.9
43.7
48.3
64.1
Strong
Strong
1 (PPD 1)
6 (PPD 2)
7 (PPD 3)
8 (PPD 4)
9 (PPD 5)
12 (PPD 6)
13 (PPD 7)
15 (PPD 8)
17 (PPD 9)
20 (PPD 10)
Moderate
Moderate
Moderate
Moderate
Moderate
Weak-moderate
Moderate
Weak-moderate
Moderate
Strong
10 21 (PPD 11)
11 28 (PPD 12)
12 29 (PPD 13)
14 Bandrowski’s Base
13 ME-PPD
14 PTD
15 Control (2,4 dinitro 1- 83.7
chloro benzene (DNCB))
10.2
8.1
10.9
Weak
Weak-moderate
Moderate
Moderate
Strong
6.9
24.8
25.3
44.6
Strong
16 Control
(Cinnamaldehyde)
17 Control (Lactic acid)
35.4
12.7
0.3
24.0
2.5
Moderate
Weak
4.8
The two model heptapeptide-containing nucleophilic amino acids PPD 2-5, PPD 12 and PPD 13) or long aliphatic chain at the one end
cysteine and lysine are used in the DPRA to simulate the protein of the amine functionality (as in PPD 9): these substitutions made
reactivity event within the skin sensitization adverse outcome PPD amine unavailable for the electronic oxidation, which
pathway. OECD guidelines recommend the use of mean cysteine minimizes the formation of reactive imines and, thus, becomes less
and
reactive towards peptides in comparison to PPD.
lysine percent peptide depletion values as a way of discriminating Among the synthesized derivatives, PPD 12 and PPD 13 showed the
between skin sensitizers and non-sensitizers.20 The average peptide lowest mean peptide depletion, with values of 17.9% and 23.0%,
reactivities determined for 24 h place the derivatives in the weakly respectively, suggesting the least expected side effects in the whole
or moderately reactive categories (Table 1).
PPD derivatives series. In both derivatives, one end of the amine
They display lower reactivity compared to PPD, ME-PPD and PTD functionality was blocked by a benzyl substituent, which resulted in
due to suitable substituents possessing different electronic the unavailability of the amine for oxidation to form the imine
properties. PPD undergoes auto-oxidation to form an electrophilic conjugate intermediate, and this could have contributed to the
imine intermediate21 that is reactive to nucleophilic amino acids. lower extent of binding with the model peptides. PPD 6, 7 and 9
Similarly, for our PPD derivatives to be reactive towards showed depletion values of 27.1%, 24.1 and 23.9%, respectively, i.e.
nucleophilic amino acids, such an imine intermediate is expected to 2 times less binding than PPD. These derivatives have strong
form. However, by modifying and substituting the PPD at the ortho electron donating substituents at the ortho position of PPD. This
position (as in PPD 6-9) with stronger electron donating chain than could render the benzene ring electron rich by electron donating
PTD (ortho methyl) and ME-PPD (ortho methoxy methyl), the resonance effect, making it less available for nucleophilic protein
electron density of the benzene ring is expected to increase through interaction. A lower value of mean depletion observed for PPD 7 vs.
electron donating resonance effects of the ortho O-alkyl functional PPD 6 was likely due to a stronger donating effect of branched ethyl
chain. Stronger donating chain like O-hexyl increase electron hexyl chain in PPD 7. Conversely, the lower depletion of PPD 9 was
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due to an additional N-hexyl substitution, which made the
compound less susceptible to oxidation than PPD 6 by blocking the
amine functionality, in addition to making the system less
nucleophilic with electron donating resonance effect of the O-hexyl biological activities of test compounds based on peptide
chain.
depletion.20 However, it is not known
View Article Online
DOI: 10.1039/C9NJ03362A
Table 1. DPRA classification results of PPD derivatives
The other derivatives (PPD 1, 10 and 11) displayed high mean if and what kind of reactions to living cells this DPRA test is
peptide depletion values. These molecules are dimeric, trimeric and predicting. It seems that it can predict to a certain extent the
tetrameric structures of PPD and are un-substituted. They are binding and interaction of chemical structures to skin cells. This
expected to form electrophilic conjugated imine intermediates, e.g. interaction can induce damage to skin cells in the form of irritant
indamine dye in case of dimeric PPD 1 and emeraldine dye-like dermatitis and eventual allergic dermatitis through release of
compound in case of PPD 10 and 11 upon auto oxidation, as cytokines and proliferation of the slightly damaged skin cells.22
benzoquinone diamine (BQDI) observed for PPD. Hence, it is not
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surprizing that these derivatives naturally have higher tendency to Hair dyeing
make binding with nucleophiles of tested peptides. Interestingly, Figure 2 shows the hair dyeing results obtained from PPD, PPD 1, 6,
the dimeric form of PPD 1 displayed higher peptide depletion than 7, 8, 10, PTD and ME-PPD applied as formulation A (aqueous
trimer PPD 10 and tetramer PPD 11. Another trimeric form of PPD, solution of derivative alone). The derivatives dyes impart a vibrant
i.e. Bandrowski’s base, showed similar mean peptide depletion color in contrast to the bleached hair tone.
(38.9%) to PPD 10 (Table 1). Interestingly, another dimeric PPD
derivative (PPD 8), with ortho hexyl substitution, showed slightly Hair coloring results of the same derivatives in formulations B (with
lower peptide depletion (34%) as compared to its unsubstituted the oxidant H2O2) and C (with the coupler resorcinol) are shown in
counterpart PPD 1 (46.6%), proving the fact that addition of the O- the Supplementary Information (Figure S15) along with the
hexyl side chain at ortho position is beneficial in decreasing the measured color tone and color difference values (Table S2). The
peptide depletion due to electron donating resonance effect.
results of the colour measurement are represented as the L, a, b
Another series of PPD derivatives, symmetrically aryl capped oligo values and further these values are converted into delta H, delta E
anilines PPD 2-5 are moderate-to-weak sensitizers according to the values using the formula presented in the supplementary section.
DPRA. Among this series of derivatives, PPD 2 displayed higher As noticed in the Figure 2, PPD, PTD and ME-PPD showed no colour
depletion (33.4%) as compared to rest of the compounds, because without oxidizing agent. Whereas the oxidized PPD, PTD and ME-
of the presence of hydrophilic hydroxyl head group of the benzyl PPD showed black or brown colour (Figure S15). It was observed
chain at both the end of the amine cap, which could be involved in that ΔH & ΔE value of PPD, PTD and ME-PPD type A is comparable
hydrogen bonding interactions with heptapeptides. Other to the bleached hair, which indicates that PPD, PTD and ME-PPD
commercially available dye precursors of PPD derivative such as have no intrinsic dyeing efficiency. As expected, the oxidized PPD
ME-PPD and PTD, showed a mean peptide depletion of 43.7%, 48.3 type B (upon treatment with H2O2) showed similar ΔH & ΔE value
%, respectively. This suggests that the addition of weaker electron as the original black hair, indicating that PPD oxidized product is
donating chain in ME-PPD and PTD, at the ortho position of PPD did black
not affect the electron density of the benzene ring. As expected, the
ortho substitutions in these compounds caused weak electron
donating inductive effects to the benzene ring, and made ortho and
para positions sites (such as position 3, 5 and 6) of ME-PPD and PTD
weaker nucleophiles than PPD 6, 7 and 9. Hence, these compounds
were still moderate and strong sensitizers in the DPRA assay.
All PPD derivatives tested (PPD 2-5, 6-9 and 12-13) showed
significantly lower mean peptide depletion percentages than PPD.
This corroborates the hypothesis that their structural diversity (with
increased electron density and/or protection of terminal amine by
benzyl substituents) might indeed be associated with improved
safety in cosmetic products. In fact the most promising compounds
from the DPRA assay were also shown to minimally penetrate
through the skin using porcine ear skin inserted in Franz diffusion
cells (data not shown). The results also signify the importance of
having strong electron donating substituents at the ortho position
of PPD, with large chains able to minimize the reactivity towards
peptides and potential skin-related side effects. It should be noted
that the classification according to DPRA assays is a preliminary
screening tool to estimate the potentially undesired
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View Article Online
DOI: 10.1039/C9NJ03362A
Conclusions
in colour. PTD and ME-PPD type B showed similar ΔH & ΔE value
responsible for brown colour. PPD 1 showed black colour and its ΔH
value (Table S2) is comparable to the natural hair shaft. PPD 6 type
We have synthesized and characterised novel oligomeric PPD
derivatives with functionalized side chains for use as safer, less toxic
hair dyes. Among the PPD derivatives, PPD 6 (13), PPD 7(14) and the
dimeric form of oligo aniline, PPD 8 (15), PPD 10(20), showed a
reduced sensitizing potential in the DPRA assay and stably colored
bleached hair samples without staining the skin. Although PPD 9, 12
and 13 showed weak sensitizing potential, their poor coloring
properties preclude them as hair dyes. Our work represents a major
step in the development of novel hair dyes, together with recently
reported structures that are safer than currently marketed
products. The potential for chemical structures such as hair dye
ingredients to cause sensitization depends, among other
interactions with skin, on their ability to penetrate the skin barrier
and to covalently modify epidermal proteins.
A showed intense
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PPD
(No
color)
PPD 1
(Black)
PPD 6
PPD 7
PPD 8
PPD 10
(Bluish
Purple)
(Magenta) (Purple) (Black)
Experimental Section
Materials
All the commercial materials for the synthesis was obtained from
Sigma Aldrich (Singapore) and were used without further
purification. For hair dyeing experiment, PPD (98.0%) and resorcinol
(≥ 99.0%) were purchased from Sigma-Aldrich (Singapore).
Schwarzkopf BlondMe Premium Lift 9+ bleaching powder and
Schwarzkopf BlondMe 12%/40 vol developer solution, Silkpro VitAir
series daily balance shampoo, and Silkpro VitAir series daily
treatment Masque (hair conditioner) were purchased from Amazon
and used without further purification. 30% hydrogen peroxide
solution was purchased from Merck (Singapore). For DPRA
experiment, HPLC grade ACN (99.0%), methanol (99.0%) from Fisher
Chemical (Fischer Scientific, Belgium), leucine enkephalin acetate
salt hydrate (95.0%), deferoxamine Mesylate (92.5%), lysine-
containing (Ac-RFAAKAA-COOH, 95.5%) and cysteine-containing
(Ac-RFAACAA-COOH, 96.05%) heptapeptides were purchased from
Peptide 2.0 Inc (Chantilly, VA). All other chemicals were purchased
from Sigma Aldrich, Singapore. 1,4-dithiothreitol (DTT) (98.0%),
monobasic sodium phosphate (99.0%), dibasic sodium phosphate
(98.5%), ammonium acetate (98.0%), cinnamaldehyde (98.0%), 2,4
dinitro-1-chloro benzene (DNCB) (97.0%), ethyl acrylate (99.0%),
glutaraldehyde (50 wt % in H2O), lactic acid (97.5%), lactose
(98.0%), methyl salicylate (> 99.0%), 3-methylcatechol (98.0%) were
purchased from Sigma-Aldrich (Singapore).
PTD
ME-PPD
(No color) (No color)
Figure 2. Hair colors obtained from PPD derivatives 1, 6, 7, 8, 10,
PTD and ME-PPD in aqueous solution (formulation A).
magenta colour with high ΔE, ΔH value and the oxidation type
displayed black colour nuance. PPD 7 displayed a purple colour with
higher ΔH & ΔE value higher than PPD 6. PPD 8 displayed black
colour for both types A & B, which resembled the natural black hair.
PPD 10 showed higher ΔH value for all 3 types indicating grey colour
dye.
Our hair dye experiments confirmed that our PPD derivatives were
intrinsically colored and able to provide hair nuance even in the
absence of the in situ oxidation with a developer (i.e. strong
oxidizing agent). This interesting result, although confined to
bleached hair samples, indicates that our PPD derivatives can in
principle avoid the usage of oxidizers like H2O2, which is known to
damage the hair cuticle.23,24 Moreover, the color on the hair was
stable for several weeks upon repeated washes with shampoo and
conditioner (Table S3), indicating that our PPD derivatives were
“locked” stably in the form of permanent hair dyes. It could be
argued that traces of H2O2 could have been responsible for this
observation, but this hypothesis was rejected as it would have
shown a black color for PPD; instead it remained colorless during
the first 30 minutes of application (Figure 2, PPD, PTD and ME-PPD
samples). The presence of the dyes on the hair was also confirmed
by the quantitative evaluation of hair samples through LC-MS/MS
and UV spectroscopy, confirming that the dyeing compounds are
retained on the hair over time (data not shown). This opens up the
possibility for PPD 1, 6, 7, 8 and 10 to be incorporated into hair dye
preparations to provide persistent and bright colors to hair.
Instrumentation
NMR spectra were recorded on a Bruker Ultrashield 300MHz
system, Bruker Ultrashield Plus 400MHz system and Bruker
Ultrashield 500MHz system, at room temperature with DMSO-d6 as
the solvent and TMS as the internal standard. H and 13C chemical
1
shifts are reported in ppm downfield of tetramethylsilane with
reference to the residue solvent peak. Coupling constant (J) were
reported in Hertz (Hz) and signal couplings are reported using the
following abbreviations: s, singlet; d, doublet; t, triplet; m,
multiplet; p, pentet; q, quartet; br, broad resonance. High
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resolution mass spectra (HRMS) were obtained on a Finnigan/MAT 4-[(E)-(4-methoxyphenyl)methyleneamino]-N-[4-[(E)-(4-methoxy
View Article Online
DOI: 10.1039/C9NJ03362A
95XL-T spectrometer system equipped with electrospray ionization phenyl) methylene-amino]phenyl]aniline (4)
(ESI) and, or Atmospheric Pressure Chemical Ionization (APCI) Using the reagents in Scheme 1 and general procedure A, the title
mode.
compound, was isolated as a golden yellow crystalline powder.
Yield: 0.36 g (84.0%). H NMR (400 MHz, DMSO-d6): δ 8.57 (s, 2H,),
8.34 (s, 1H), 7.86 (d, J = 8 Hz, 4H), 7.24 (d, J = 8 Hz, 4H), 7.12 (d, J = 8
Hz, 4H), 7.06 (d, J = 8 Hz, 4H), 3.83 (s, 6H). 13C NMR (100 MHz,
DMSO-d6): δ 161.4, 156.4, 143.5, 141.6, 129.9, 129.4, 122.2, 117.2,
114.2, 55.3.
1
4,4′-dinitrodiphenylamine
Solid sodium hydroxide (1.65 g, 41 mmol) was added to a DMSO
solution of p-nitroaniline (1.4 g, 10 mmol). To the greenish brown
mixture, nitrobenzene (3.8 mL, 37 mmol) was added dropwise and
the mixture was stirred at 80°C temperature for 12 hours with a
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slow stream of air bubbling through. After complete consumption 4-[(E)-p-tolylmethyleneamino]-N-[4-[(E)-p-tolylmethylene amino]
of nitrobenzene, as evident from TLC analysis, the reaction mixture phenyl]aniline (5)
was cooled to room temperature and was added dropwise to water Using the reagents in Scheme 1 and general procedure A, the title
with vigorous stirring, which resulted in a yellow precipitate. The compound was isolated as a golden yellow crystalline powder.
1
precipitate was collected by filtration, washed thoroughly with Yield: 0.32 g (80%). H NMR (400 MHz, DMSO-d6): δ 8.62 (s, 2H),
water to remove traces of NaOH and then with hexane to remove 8.40 (s, 1H), 7.81 (d, 4H, J = 8 Hz), 7.32 (d, 4H, J = 8 Hz), 7.28 (d, 4H,
excess nitrobenzene. The precipitate was dissolved in minimum J = 8 Hz), 7.13 (d, 4H, J = 8 Hz), 2.38 (s, 6H). 13C NMR (100 MHz,
amount of DCM/acetone mixture and added dropwise to hexane DMSO-d6): δ 157.3, 143.8, 142.3, 142.3, 134.5, 129.8, 128.8, 122.8,
with vigorous stirring. Yellow precipitate was filtered, washed with 117.5,21.6.
hexane and dried under suction to provide 2 g of pure product.
Yield: 2.0 g (76.6%). H NMR (500 MHz, DMSO-d6): δ 9.98 (s, 1H),
8.21 (d, J = 9 Hz, 4H), 7.36 (d, J = 9 Hz, 4H). 13C NMR (125 MHz,
DMSO-d6): δ 147.6, 140.5, 125.8, 117.1.
1
Synthesis of symmetrically amine capped oligoanilines (1). 4,4′-
Dinitrodiphenylamine (1) (PPD 1) (1 g, 3.86 mmol) was suspended in
20 mL of concentrated hydrochloric acid under nitrogen
atmosphere. Fine tin powder (2.46 g, 20.72 mmol) was added
General method for the synthesis of imines of 4,4’- portion wise to the reaction mixture. After complete addition of tin,
diaminodiphenylamine (2-5) the reaction mixture was refluxed for 18 h. The reaction mixture
General procedure. Substituted aromatic aldehyde (1.1 mmol) and was cooled to room temperature and diluted with 50 mL of water.
p-toluene sulfonic acid (5 mol %) were added to an ethanolic The pH of the mixture was set at 12 by dropwise addition of 20%
suspension of 4,4’-diaminodiphenylamine (1 mmol). Resulting aqueous NaOH solution. Greyish white precipitate formed and was
mixture was stirred at room temperature overnight. Resulting filtered and washed with water to get rid of traces of NaOH. After
crystalline precipitate was filtered and washed with a 1:1 mixture of prolonged drying the product was collected as greyish white
ethanol and diethyl ether and then finally with diethyl ether. Drying powder. Further, this compound was dissolved in 2 mL of methanol,
under suction afforded the desired product as crystalline solid. Pure to which 0.5 mL of conc. HCl was added dropwise slowly over 20
imine was obtained by the recrystallization from a mixture of min and stirred at 0 °C for 6 h. The title product was precipitated
dichloromethane and hexane.
out as hydrochloride salt. The spectral data is consistent with
literature data.19 Yield: 0.6 g (78.1%); Grey powder, decompose at
220.5°C (±0.1). 1H NMR (400 MHz, DMSO-d6): δ 6.74 (s, 1H), 6.66 (d,
J = 6.8 Hz, 4H), 6.47 (d, J = 8.8 Hz, 4H), 4.47 (s, 4H). 13C NMR (100
MHz, DMSO-d6): δ 141.4, 135.6, 118.5, 1s15.0. HRMS (ESI): calcd
for C12H14N3 [M+H]+: 200.1182, found: 200.1185.
4-[(E)-[4-[4-[(E)-(4-hydroxyphenyl)methyleneamino]anilino]
phenyl]iminomethyl] phenol (2)
Using the reagents in Scheme 1 and general procedure A, the title
compound, was isolated as a yellow-orange crystalline powder.
1
Yield: 0.3 g (75%). H NMR (400 MHz, DMSO-d6): δ 10.03 (s, 2H),
8.51 (s, 2H), 8.31 (s, 1H), 7.75 (d, J = 8 Hz, 4H), 7.25 (d, J = 6.5 Hz, Synthesis of symmetrically aryl capped oligoanilines (6-9).
4H), 7.11 (d, J = 7 Hz, 4H), 6.88 (d, J = 8 Hz, 4H). 13C NMR (100 MHz, General synthetic method for the reduction of imines of 4,4’-
DMSO-d6): δ 160.6, 157.2, 144.2, 142.0, 130.7, 128.4, 122.6, 117.7, diaminodiphenylamine (6-9). The imine (1 mmol) was dissolved in
116.1.
mixture of 12 mL of dry THF/MeOH in a two-necked round bottom
flask under nitrogen atmosphere. To the solution excess sodium
borohydride (4 mmol) was added and the mixture was stirred at
room temperature for 24 h. Excess water was added to quench the
reaction as well as to quench any unreacted sodium borohydride.
THF was removed under vacuum and the residue was extracted
with dichloromethane. The DCM extract was washed thrice with
water and then with brine solution. After drying over anhydrous
Na2SO4 and removal of solvent under vacuo afforded the desired
amines as crystalline solid.
4-[(E)-(4-nitrophenyl)methyleneamino]-N-[4-[(E)-(4-nitrophenyl)
methyleneamino] phenyl] aniline (3)
Using the reagents in Scheme 1 and general procedure A, the title
compound was isolated as a green powder.
1
Yield: 0.44 g (95%). H NMR (400 MHz, DMSO-d6): δ 8.87 (s, 2H),
8.71 (s, 1H), 8.36 (d, J = 8 Hz, 4H), 8.16 (d, J = 8 Hz, 4H), 7.42 (d, J = 8
Hz, 4H), 7.20 (d, J = 8 Hz, 4H). 13C NMR (100 MHz, DMSO-d6): δ
155.1,148.8, 143.1, 142.8, 142.7, 129.6, 124.5, 123.6, 117.8.
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4,4'-(((azanediylbis(4,1 phenylene))bis(azanediyl))Bis(methylene)) Yield: 3.2 g (67.3%). 1H NMR (300 MHz, DMSO-d6) δ 7.72 (dd, J = 8.8
View Article Online
DOI: 10.1039/C9NJ03362A
diphenol (6). The title compound was synthesized from the Hz, 2.4 Hz, 1H), 7.55 (d, J = 2.4 Hz, 1H), 6.66 (d, J = 8.8 Hz, 1H), 6.34
corresponding imine using general procedure. Yield: 0.27 g (67%): (s, 2H), 4.04 (t, J = 6.4 Hz, 2H), 1.75 (p, J = 7 Hz, 2H), 1.52-1.39 (m,
Red powder of mp 145.4-146.4°C (±0.1-0.2). 1H NMR (400 MHz, 2H), 1.31 (m, 4H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO)
DMSO-d6): δ 9.21 (s, 2H), 7.15 (d, J = 8 Hz, 2H), 6.80 (s, 1H), 6.73- δ 146.4, 144.2, 136.0, 119.9, 111.2, 106.7, 68.6, 31.4, 28.8, 25.4,
6.67 (m, 9H), 6.47 (d, J = 8.8 Hz, 4H), 5.45 (s, 2H), 4.05 (d, J = 4.0 Hz, 22.4, 14.2.
5
6
7
8
4H). 13C NMR (100 MHz, DMSO-d6): δ 156.0, 142.3, 135.4, 130.6,
9
2-(2-ethylhexyloxy)-4-nitroaniline (11)
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128.5, 122.1, 118.3, 114.9, 113.4, 47.0. HRMS (ESI): calcd for
C26H25N3O2 [M+H]+: 411.1941 found: 411.1931.
2-amino-5-nitrophenol (1.54 g, 10 mmol) was dissolved in 20 mL of
DMF kept under an inert atmosphere and 1.39 g (10 mmol) was
N1-(4-nitrobenzyl)-N4-(4-((4-nitrobenzyl) amino)phenyl) benzene- added to the stirring solution. After five minutes of stirring at room
1,4-diamine (7). Yield: 0.37 g (82%). Reddish brown powder of mp temperature, 1-bromo-2-ethylhexane (2.3 ml, 12.93 mmol) was
153.1-155.1°C (±0.1-0.3). 1H NMR (400 MHz, DMSO-d6): δ 8.18 (d, J added. Resulting red mixture was refluxed for 18 hours. The dark
= 8.8 Hz, 4H), 7.61 (d, J = 8.8 Hz, 4H), 6.87 (s, 1H), 6.67 (d, J = 8.8 Hz, brown mixture was added dropwise to saturated NaHCO3 solution
4H), 6.43 (d, J = 8.8 Hz, 4H), 5.91 (s, 2H), 4.34 (s, 4H). 13C NMR (100 with vigorous stirring. After 30 minutes the mixture was extracted
MHz, DMSO-d6): δ 149.6, 146.3, 141.5, 135.7, 128.1, 123.4, 118.4, with DCM and the organic layer was washed 3 times with NaHCO3
113.4, 46.7. HRMS (ESI): calcd for C26H23N5O4 [M+H]+: 469.1745, solution, 3 times with saturated LiCl solution and finally with brine
found: 469.1747.
solution. After drying over anhydrous Na2SO4, solvent was removed
under reduced pressure and dried under vacuo. The title compound
was isolated as yellowish-green viscous liquid.
N1-(4-methoxybenzyl)-N4-(4-((4-methoxybenzyl)amino)
)benzene-1,4-diamine (8). Yield: 0.38 g (88%). Golden brown
powder of mp 160.0-162.0°C (±0.4-0.5). H NMR (400 MHz, DMSO-
d6): δ 7.27 (d, J = 8.8 Hz, 4H), 6.87 (d, J = 8.8 Hz, 4H), 6.81 (s, 1H),
6.67 (br, 4H), 6.47 (d, J = 7.6 Hz, 4H), 5.54 (s, 2H), 4.11 (s, 4H), 3.72
(s, 6H). 13C NMR (100 MHz, DMSO-d6): δ 158.0, 142.2, 135.5, 132.5,
128.4, 118.3, 113.6, 113.4, 55.0, 46.8. HRMS (ESI): calcd for
C28H29N3O2 [M+H]+: 439.2254,found: 439.2253.
phenyl
1
Yield: 0.8g (52%). H NMR (300 MHz, DMSO-d6) δ 7.72 (dd, J = 8.8
1
Hz, 2.2 Hz, 1H), 7.57 (d, J = 2.3 Hz, 1H), 6.67 (d, J = 8.8 Hz, 1H), 6.30
(s, 2H), 3.94 (d, J = 5.3 Hz, 2H), 1.74 (dt, J = 12.0 Hz, 5.9 Hz, 1H),
1.53-1.38 (m, 4H), 1.33-1.26 (m, 4H), 0.92-0.85 (m, 6H).
2-hexyloxy-1,4-diaminobenzene (12). Excess tin(II) chloride (7.14 g,
37.6 mmol) was added to an ethanolic solution of 1.31 g (5.5 mmol)
of 2-hexyloxy-4-nitroaniline under inert atmosphere. Concentrated
HCl (2.5 ml) was added and the mixture was refluxed for 24 hours.
The resulting off white cloudy mixture was poured into ice-water
mixture and the pH of the mixture was set at 12 with 20% aqueous
NaOH solution. The mixture was extracted with ethyl acetate and
the organic layer was quickly washed with water and finally with
brine solution. After solvent evaporation, the crude residue was
purified by column chromatography eluting with ethylacetate and
petroleum ether (3:7) to give a purple residue. Further, this residue
was dissolved in 2 mL of methanol, to which 0.5 mL of conc. HCl
was added dropwise slowly over 20 min and stirred at 0 °C for 6 h.
The title product was precipitated out as hydrochloride salt.
N4-(4-tolylmethyl)-N1-[4-(p-tolylmethylamino) phenyl] benzene-
1,4-diamine (9). Yield: 0.36 g (90%).: Reddish brown powder of mp
155.5-157.5°C (±0.2-0.3). 1H NMR (400 MHz, DMSO-d6): δ 7.24 (d, J
= 8.0 Hz, 4H), 7.11 (d, J =8.0 Hz, 4H), 6.81 (s, 1H), 6.67 (br, 4H), 6.46
(d, J = 7.2 Hz, 4H), 5.59 (s, 2H), 4.14 (s, 4H), 2.27 (s, 6H). 13C NMR
(100 MHz, DMSO-d6): δ 144.3, 137.4, 135.5, 135.4, 128.7, 127.2,
122.2, 113.1, 46.7, 20.6. HRMS (ESI): calcd for C28H29N3 [M+H]+:
407.2356, found: 407.2363.
Synthesis of C-substituted PPD derivatives (12-13 &15).
2-hexyloxy-4-nitroaniline (10)
Yield: 0.96 g (84%).: purple powder decompose at 204.2°C (±0.2). 1H
NMR (400 MHz, DMSO-d6) δ 6.38 (d, J = 8.0 Hz, 1H), 6.18 (d, J = 2.0
Hz, 1H), 5.98 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 4.04 (br, 4H), 3.83 (t, J = 6.4
Hz, 2H), 1.73 – 1.66 (m, 2H), 1.44 – 1.40 (m, 2H), 1.32-1.30 (m, 4H),
0.88 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 146.7,
139.9, 127.6, 115.4, 106.6, 100.4, 67.4, 31.0, 28.9, 25.2, 22.1, 13.9.
HRMS (ESI): calcd for C12H21N2O [M+H]+: 209.1648, found: 209.1650.
2-Amino-5-nitrophenol (3.08 g, 20 mmol) was dissolved in 10 mL of
DMF kept under an inert atmosphere and 1-bromohexane (2.87 ml,
20.5 mmol) and K2CO3 (2.83 g, 20.5 mmol) were added to the
solution. The resulting red mixture was refluxed for 18 hours, after
which the resulting dark brown mixture was added dropwise to
saturated NaHCO3 solution with vigorous stirring. After 30 minutes,
the mixture was extracted with DCM and the organic layer was
washed 3 times with NaHCO3 solution, 3 times with saturated LiCl
solution and finally with brine solution. After drying over anhydrous
Na2SO4, the solvent was removed under reduced pressure and the
resulting mixture oil dried in vacuo. The oily mixture was then
purified by gradient column chromatography with ethyl acetate and
hexane mixture as eluent. The compound was eluted with 50%
ethyl acetate and hexane mixture and solvent evaporation afforded
the product as a bright yellow solid.
2-((2-ethylhexyl)oxy)benzene-1,4-diamine (13). The title product
was synthesized same manner as the procedure described for 2-
hexyloxy-1,4-diaminobenzene (12). Yield: 0.6 g (82%).: pale purple
1
powder of melting point 225-226°C (±0.2-0.3). H NMR (400 MHz,
DMSO-d6) δ 6.38 (d, J = 8.0 Hz,1H), 6.20 (d, J = 2.4Hz, 1H), 5.98 (dd,
J = 8.0 Hz, 2.0 Hz, 1H), 4.04 (s, 4H), 3.73 (d, J = 5.6 Hz, 2H), 1.70-1.65
(m, 2H), 1.47-1.39 (m, 4H), 1.38 -1.29 (m, 4H), 0.88 (t, J = 6.0 Hz,
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6H). 13C NMR (100 MHz, DMSO-d6) δ 146.9, 139.9, 127.5, 115.3, 2 mL of conc. HCl to give title compound as black solid. Yield: 0.94 g
View Article Online
1
DOI: 10.1039/C9NJ03362A
106.5, 100.2, 69.8, 30.0, 28.5, 23.4, 22.5, 13.9, 10.9. HRMS (ESI): (99.0%).: black solid of mp 161.2-162.3°C (±0.1-0.3). H NMR (400
calcd for C14H25N2O [M+H]+: 237.1961, found: 237.1958.
MHz, DMSO-d6) δ 6.30 (d, J = 8.4 Hz, 1H), 6.21 (d, J = 2.4 Hz, 1H),
6.05 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 4.28 (br, 2H), 3.84 (t, J = 6.4 Hz, 2H),
2.92 (t, J = 6.7 Hz, 2H), 1.72 - 1.65 (m, 2H), 1.52 -1.45 (m, 2H), 1.42-
1.39 (m, 3H), 1.31-1.26 (m, 10H), 0.86 (q, J = 7.0 Hz, 6H). 13C NMR
(100 MHz, DMSO-d6) δ 146.9, 139.5, 129.5, 111.6, 106.4, 100.3,
67.6, 44.3, 31.1, 31.0, 29.0, 28.8, 26.3, 25.3, 22.1, 22.0, 13.8. HRMS
(APCI): calcd for C16H25N2O3 [M+H]+: 293.2587, found: 293.2587.
4-Amino-(3-hexyloxy)-4′-nitrodiphenylamine (14)
Triethyl amine (0.635 ml, 4.6mmol, 1.5 equivalent) was added to a
DMSO (7 mL) solution of the equimolar mixture of 1-Fluoro-4-
nitrobenzene (0.323 ml, 3.04 mmol) and 2-hexyloxy-1,4-
diaminobenzene (0.63 g, 3.04 mmol). After 24 hours of stirring at
90°C, reaction mixture was cooled and poured into water to induce
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precipitation. The sticky precipitate was extracted with ethyl Synthesis of symmetrically amine (NH2/NH2) capped oligoanilines
acetate and the organic layer was washed thoroughly with water (20) and (21).
and brine solution. Evaporation of solvent afforded the crude
product as red liquid which was purified by silica gel column
N1,N4-bis(4-nitrophenyl)- 1,4-benzenediamine (18)
1-Fluoro-4-nitrobenzene (2.2 mL, 20.7 mmol) and triethylamine (3
mL, 21.5 mmol) was added successively to 20 ml DMSO solution of
p-phenylenediamine (1.08 g, 10 mmol) under inert atmosphere. The
dark red solution was stirred at 90°C for three days, after which the
mixture was cooled to room temperature and was dropwise added
to 200 ml of chilled water with vigorous stirring. Dark brown
precipitate was collected by filtration and washed thoroughly with
distilled water. Drying under suction afforded the crude product
chromatography.
Yield: 0.89 g (89%).1H NMR (300 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.00
(d, J = 9.2, 2H), 6.81 (d, J = 9.3, 2H), 6.71 – 6.64 (m, 2H), 6.59 (dd, J =
8.3, 1.9, 1H), 4.66 (s, 2H), 3.92 (t, J = 6.4, 2H), 1.77 – 1.67 (m, 2H),
1.42 (dd, J = 13.2, 6.0, 2H), 1.34 – 1.26 (m, 4H), 0.87 (t, J = 6.8, 3H).
13C NMR (75 MHz, DMSO-d6) δ 153.3, 146.3, 136.8, 135.9, 128.8,
126.6, 116.4, 114.3, 112.3, 108.3, 68.2, 31.4, 29.1, 25.6, 22.4, 14.2.
4-Amino-(3-hexyloxy)-4′-aminodiphenylamine (15). The title which was purified by silica gel column chromatography using 20%
product was synthesized by same manner as the procedure acetone/DCM mixture to afford dark green solid. Alternatively, the
described for 2-hexyloxy-1,4-diaminobenzene (12). Yield: 0.85 g product can be isolated by washing the crude mixture with 70%
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(85%).: dark green powder decompose at 182.5°C (±0.5). H NMR DCM/hexane mixture. The spectral data is consistent with literature
(400 MHz, DMSO-d6) δ 6.66 (d,8.0 Hz 1H), 6.61 (d, J = 8.8 Hz, 2H), data. 19
6.43 (d, J = 8.4 Hz, 2H), 6.27 (d, J = 2.4, 1H), 4.58 (s, 2H), 4.45 (s, 2H), Yield: 1.40 g (40%). H NMR (500 MHz, DMSO-d6): δ 9.31 (s, 2H),
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3.85 (t, J = 6.4 Hz, 2H), 1.70 – 1.64 (m, 2H), 1.40 – 1.33 (m, 2H), 8.10 (d, J = 9.3 Hz, 4H), 7.28 (s, 4H), 7.04 (d, J = 9.3 Hz, 4H). 13C NMR
1.28-1.23 (m, 4H), 0.87 (t, J = 4.8 Hz, 3H). 13C NMR (100 MHz, (125 MHz, DMSO-d6): δ 150.0, 137.6, 135.6, 126.2, 122.4, 113.0.
DMSO-d6) δ 149.8, 143.6, 131.6, 125.1, 123.9, 122.9, 118.5, 117.3,
4,4'-Bis[(4-nitrophenyl)amino]diphenylamine (19)
115.4, 108.1, 68.5, 31.0, 28.4, 25.0, 22.0, 13.9. HRMS (ESI): calcd for
C18H26N3O [M+H]+: 300.2070, found: 300.2073.
1-Fluoro-4-nitrobenzene (0.8 mL, 7.5 mmol) and triethylamine (1
mL, 7.2 mmol) was added successively to 7 ml DMSO solution of
4,4′-diaminodiphenylamine (0.497 g, 2.5 mmol) under inert
atmosphere. The dark red solution was stirred at 90°C for three
days, after which the mixture was cooled to room temperature and
was dropwise added to 200 ml of chilled water with vigorous
stirring. Dark brown sticky precipitate was extracted with methanol
and the supernatant with DCM. Combined extract was evaporated
to dryness and the black sticky residue was dissolved in DMF and
triturated with a 1:1 mixture of Et2O and hexane to remove excess
1-fluoro-4-nitrobenzene. Further repeated trituration from DCM
and hexane afforded the title compound as dark brown crystalline
solid. The spectral data is consistent with literature data. 19
Synthesis of C,N-substituted PPD derivatives (17).
(N1-hexyl)(2-hexyloxy)-4-nitroaniline (16)
The title compound was obtained by analogy to the process to
make 2-hexyloxy-4-nitroaniline (10) above, except that 41 mmol of
1-bromohexane and K2CO3 were used.
The compound was isolated from the reaction mixture by elution
with a 20% ethyl acetate and hexane mixture. A bright yellow
viscous liquid was obtained upon evaporation of the effluents.
Yield: 4.90 g (76%). 1H NMR (300 MHz, DMSO) δ 7.81 (dd, J = 9.0 Hz,
2.4 Hz, 1H), 7.53 (d, J = 2.5 Hz, 1H), 6.61 (d, J = 9.0 Hz, 1H), 6.35 (t, J
= 5.4 Hz, 1H), 4.07 (t, J = 6.4 Hz, 2H), 3.23 (dd, J = 13.6 Hz, 6.6 Hz,
2H), 1.82 – 1.70 (m, 2H), 1.54 (m, 2H), 1.44 (m, 2H), 1.38-1.23 (m,
10H), 0.87 (q, J = 6.8 Hz, 6H). 13C NMR (75 MHz, DMSO) δ 145.3,
144.4, 135.4, 120.2, 106.8, 105.6, 68.8, 42.43, 31.3, 28.7, 28.6, 26.4,
25.5, 22.4, 14.2.
1
Yield: 0.96 g (87%). H NMR (500 MHz, DMSO-d6): δ 9.13 (s, 2H),
8.23 (s, 1H), 8.06 (d, J = 9.3 Hz, 4H), 7.14 (q, J = 8.9 Hz, 8H), 6.92 (d, J
= 9.3 Hz, 4H). 13C NMR (75 MHz, DMSO-d6): δ 152.0, 140.3, 137.0,
131.7, 126.2, 123.6, 117.6, 112.3.
N1,N4-bis(4-aminophenyl)-1,4-benzenediamine
(20).
This
(N1-hexyl)(2-hexyloxy)- 1,4-diaminobenzene (17). Following the
method for the synthesis of 2-hexyloxy-1,4-diaminobenzene (12),
reduction of (N1-hexyl)(2-hexyloxy)-4-nitroaniline (1.05 g, 3.1 mmol)
was performed using 3 g (15.8 mmol) tin(II) chloride in presence of
compound was synthesized by the reduction of corresponding
dinitro compound N1,N4-bis(4-nitrophenyl)- 1,4-benzenediamine
(18) (0.6 g, 1.7 1 mmol) with tin powder (2 g, 16 mmol) following
the method described for the synthesis of 4,4′-
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diaminodiphenylamine (section 1.2) to give title product as purple (E)-N1-(4-methylbenzylidene)-N4-(4-nitrophenyl)benzVeienwe-A1rt,ic4le Online
DOI: 10.1039/C9NJ03362A
oil. Further, this oil was dissolved in 2 mL of methanol, to which 0.5 diamine (24)
mL of conc. HCl was added dropwise slowly over 20 min and stirred p-Toluenecarbox aldehyde (0.39 mL, 3.27 mmol) and 5 mol% of p-
at 0°C for
6 h. The title product was precipitated out as toluenesulphonic acid was added to an ethanolic solution (10 mL) of
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hydrochloride salt. The spectral data is consistent with literature 4-amino-4′-nitrodiphenylamine (0.682 g, 2.97 mmol) and the
data.19 Yield: 0.3 g (61%).: purple solid of mp >300°C. 1H NMR (400 mixture was stirred overnight at room temperature under nitrogen
MHz, DMSO-d6): δ 6.99 (s, 2H), 6.73-6.71 (m, 8H), 6.48 (d, J = 8.3 atmosphere. Resulting orange precipitate was collected by filtration
Hz, 4H), 4.56 (s, 4H). 13C NMR (100 MHz, DMSO-d6): δ 142.1, 138.1, and washed with 20 mL of 20% ethanol/hexane mixture and finally
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134.5, 119.7, 117.1, 115.1. HRMS (ESI): calcd for C18H18N4 [M+H]+: with hexane. Drying over suction afforded pure crystalline solid.
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290.1526, found: 290.1518.
Yield: 0.97 g (98%). H NMR (300 MHz, DMSO-d6): δ 9.37 (s, 1H),
8.62 (s, 1H), 8.10 (d, J = 9.0 Hz, 2H), 7.83 (d, J = 7.9 Hz, 2H), 7.36 –
7.26 (m, 6H), 7.07 (d, J = 9.1 Hz, 2H), 2.38 (s, 3H). 13C NMR (75 MHz,
DMSO-d6): δ 159.1, 150.6, 146.6, 141.3, 138.1, 137.9, 133.6, 129.3,
128.5, 126.1, 122.2, 121.5, 113.4, 21.1.
N1-(4-aminophenyl)-N4-(4-nitrophenyl)-1,4-benzenediamine (21)
The title compound was obtained by analogy to the process to
make compound 20 above, except that 4,4′-diaminodiphenylamine
(0.5948 g, 2.98 mmol) was used instead of p-phenylenediamine.
Black solid. Yield: 0.89 g (65.8%). H NMR (300 MHz, DMSO-d6): δ N1-[(4-imino-p-tolyl)
phenyl]-N4-(4-nitrophenyl)-1,4
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8.98 (s, 1H), 8.01 (d, J = 9.1 Hz, 2H), 7.53 (s, 1H), 7.01 (d, J = 8.6 Hz, benzenediamine (25)
2H), 6.83 (dd, J = 11.7 Hz, 4.9 Hz, 6H), 6.55 (d, J = 8.5 Hz, 2H), 4.76 p-Toluenecarboxaldehyde (0.11 mL, 0.96 mmol) and 5 mol% of p-
(s, 2H). 13C NMR (75 MHz, DMSO-d6): δ 152.7, 144.0, 143.8, 136.6, toluenesulphonic acid was added to an ethanolic solution (10 mL) of
131.7, 129.1, 124.3, 122.3, 114.7, 114.4, 111.9.
N1-(4-aminophenyl)-N4-(4-nitrophenyl)-1,4-benzenediamine (0.278
g, 0.87 mmol) and the mixture was stirred overnight at room
temperature under nitrogen atmosphere. Resulting orange
precipitate was collected by filtration and washed with 20 mL of
20% ethanol/hexane mixture and finally with hexane. Drying over
suction afforded pure crystalline solid.
Synthesis of unsymmetrically aryl(Ph/NH2) capped oligoanilines
(28) and (29).
4-Amino-4′-nitrodiphenylamine (22)
Excess potassium carbonate (2.8 g, 20 mmol) was added to a DMSO
(10 mL) solution of p-phenylenediamine (1.087g, 10 mmol), 1-
fluoro-4-nitrobenzene (1.1 mL, 10.4 mmol) and kept under inert
atmosphere. The dark red solution was stirred at 90°C for 3 days,
after which the mixture was cooled to room temperature and was
dropwise added to 200 mL of chilled water with vigorous stirring.
Resulting red precipitate was collected by filtration. Washing of the
filter cake with water and drying under suction afforded the crude
product which was purified by silica gel column chromatography.
The title compound was eluted with 5% ethyl acetate/DCM mixture.
1
Yield: 0.31 g (86%). H NMR (300 MHz, DMSO-d6): δ 9.13 (s, 1H),
8.61 (s, 1H), 8.31 (s, 1H), 8.06 (d, J = 9.2 Hz, 2H), 7.80 (d, J = 8.0 Hz,
2H), 7.29 (dd, J = 13.4 Hz, 8.4 Hz, 4H), 7.17 – 7.08 (m, 6H), 6.93 (d, J
= 9.2 Hz, 2H), 2.37 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 156.6,
151.9, 143.0, 142.2, 140.7, 139.8, 137.0, 133.9, 131.9, 129.3, 128.2,
126.2, 123.5, 122.3, 118.0, 116.7, 112.4, 21.05.
4-Nitro-4′-[(4-tolylaminomethyl)phenyl]diphenylamine (26)
Excess sodium borohydride (0.25 g, 6.61 mmol) was added to a
solution of the imine (0.45 g, 1.36 mmol) in a 2:1 mixture of THF
and methanol. The mixture was stirred at room temperature for 24
hours before quenching with water. Volatiles were removed under
reduced pressure and the solution pH was adjusted to 9 with
careful addition of conc. HCl and the mixture was extracted with
DCM. The organic layer was washed with water and then with brine
solution, finally dried over anhydrous Na2SO4. Solution was
concentrated and pure product was precipitated with the addition
of excess hexane. Precipitate was dried under vacuum to get brown
powder.
1
Yield: 0.99 g (43%). H NMR (300 MHz, DMSO-d6): δ 8.88 (s, 1H),
8.00 (d, J = 9.2 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 6.76 (d, J = 9.2 Hz,
2H), 6.61 (d, J = 8.5 Hz, 2H), 5.08 (s, 2H). 13C NMR (75 MHz, DMSO-
d6): δ 153.1, 146.3, 136.4, 127.8, 126.3, 124.7, 114.5, 111.7.
N1-(4-aminophenyl)-N4-(4-nitrophenyl)-1,4-benzenediamine (23)
1-Fluoro-4-nitrobenzene (0.31 mL, 2.9 mmol) and triethylamine (0.8
mL, 5.74 mmol) was added successively to 7 ml DMSO solution of
4,4′-diaminodiphenylamine (0.5948 g, 2.98 mmol) under inert
atmosphere. After 3 days of stirring at 90°C, reaction mixture was
cooled and poured into water to induce precipitation. The dark
precipitate was collected by filtration, washed with water and dried
under suction. The crude product was purified by gradient column
chromatography while the desired product eluted with 2%
DCM/ethyl acetate mixture.
Yield: 0.4 g (88%). 1H NMR (300 MHz, DMSO-d6): δ 8.90 (s, 1H), 7.99
(d, J = 9.1 Hz, 2H), 7.26 (d, J = 7.8 Hz, 2H), 7.13 (d, J = 7.8 Hz, 2H),
6.95 (d, J = 8.5 Hz, 2H), 6.76 (d, J = 9.1 Hz, 2H), 6.61 (d, J = 8.5 Hz,
2H), 6.24 (t, J = 5.9 Hz, 1H), 4.22 (d, J = 5.9 Hz, 2H), 2.27 (s, 3H). 13
C
NMR (75 MHz, DMSO-d6): δ 152.9, 146.3, 137.0, 136.4, 135.6,
128.8, 127.8, 127.1, 126.2, 124.4, 112.8, 111.7, 46.4, 20.6.
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Yield: 0.89 g (65.8%). H NMR (300 MHz, DMSO-d6): δ 8.98 (s, 1H),
N1-[4-(4-tolylaminomethyl)phenyl]-N4-(4-nitrophenyl)-1,4
benzenediamine (27)
1-Fluoro-4-nitrobenzene (0.12 mL, 1.1 mmol) and triethylamine
(0.23 mL, 1.2 mmol) was added successively to 7 mL DMSO solution
8.01 (d, J = 9.1 Hz, 2H), 7.53 (s, 1H), 7.01 (d, J = 8.6 Hz, 2H), 6.83 (dd,
J = 11.7 Hz, 4.9 Hz, 6H), 6.55 (d, J = 8.5 Hz, 2H), 4.76 (s, 2H). 13C NMR
(75 MHz, DMSO-d6): δ 152.7, 144.0, 143.8, 136.6, 131.7, 129.1,
124.3, 122.3, 114.7, 114.4, 111.9.
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of 4-amino-4′-[(4-tolylaminomethyl)phenyl]diphenylamine (28) 2H), 7.12 (d, J = 8.0 Hz, 2H), 6.87-6.75 (m, 10H), 6.51(d, J = 8.4 Hz,
View Article Online
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DOI: 10.1039/C9NJ03362A
(0.303 g, 1 mmol) under inert atmosphere. The dark red solution 4H), 5.67 (br, 1H), 4.59 (br, 2H), 4.17 (s, 2H), 2.27 (s, 3H). C NMR
was stirred at 90°C for two days, after which the mixture was (100 MHz, DMSO-d6): δ 142.7, 142.1, 138.1, 137.8, 137.6, 135.5,
cooled to room temperature and was dropwise added to 200 ml of 134.4, 128.8, 127.2, 119.7, 119.4, 117.1, 116.9, 115.0, 113.4, 47.1,
chilled water with vigorous stirring which afforded colloidal 20.7. HRMS (ESI): calcd for C26H26N4, [M+H]+: 394.2152, found:
suspension. Addition of brine solution produced dark brown 394.2157.
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precipitate which was collected by f iltration, washed with water
and dried under suction. The crude product was purified by silica
gel column chromatography by elution with pure DCM. Yield: 0.11 g
ammonium hydroxide solution (pH 9.5) and 62.5 mL of 3% H2O2
(26 %).
Synthesis of Bandrowski’s base. PPD (5g, 0.0462 mol) was
dissolved in 150 mL of water and to which 1.5 mL of 28%
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were added. The resulting reaction mixture was maintained for 24
hours at room temperature. The insoluble products were separated
Alternatively, this compound was also synthesized by reducing the
imine group of N1-[(4-imino-p-tolyl)phenyl]-N4-(4-nitrophenyl)-1,4-
benzenediamine (21) (0.2 g, 0.47 mmol) with sodium borohydride
(0.1 g, 2.64 mmol) in a 1:2 mixture of methanol/THF. Work up was
performed following the method used for the synthesis of 4-nitro-
4′-[(4-tolylaminomethyl)phenyl]diphenylamine (22) to isolate
product as red solid.
by filtration to obtain
purification, the compound was quickly dissolved in hot pyridine
and slow addition of water to this solution resulted in
a pure title compound. For further
a
precipitation of Bandrowski’s base as black solid.25 Yield: 2.2 g
(15%).: black solid of mp 236.4-237.4°C (±0.2-0.3). 1H NMR (400
MHz, DMSO-d6); 6.61-6.56 (m, 8H), 6.0 (s, 4H), 5.69 (s, 2H), 4.94 (br,
4H,), 13C NMR (100 MHz, DMSO-d6); 153.3, 148.3, 144.9, 139.9,
122.3, 114.3, 90.7; HRMS (ESI): calcd for C18H19N6 [M+H]+: 319.1666,
found: 319.1663.
1
Yield: 0.19 g (95 %). H NMR (300 MHz, DMSO-d6): δ 8.98 (s, 1H),
8.01 (d, J = 9.1 Hz, 2H), 7.55 (s, 1H), 7.26 (d, J = 7.8 Hz, 2H), 7.12 (d, J
= 7.8 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 6.90-6.80 (m, 6H), 6.56 (d, J =
8.6 Hz, 2H), 5.90 (t, J = 5.9 Hz, 1H), 4.19 (d, J = 5.8 Hz, 2H), 2.27 (s,
3H). 13C NMR (75 MHz, DMSO-d6): δ 152.6, 144.2, 143.8, 137.3,
136.6, 135.4, 131.8, 129.2, 128.7, 127.1, 126.2, 124.2, 122.1, 114.5,
113.1, 111.9, 46.8, 20.6.
Direct peptide reactivity assay
Incubation mixtures were formulated in glass auto sampler vials
using peptide to test compound ratios of both 1:10 for cysteine
peptide and 1:50 for lysine peptide.20,26,27 Stock solutions of
cysteine (Ac-RFAACAA-COOH,) and lysine (Ac-RFAAKAA-COOH,)
containing peptides was prepared fresh immediately before use.
Reactivity of PPD derivatives towards the peptides was determined
using 20 µM of cysteine and lysine in 0.1 M phosphate buffer (pH
7.5) and 0.1 M ammonium acetate buffer (pH 10.2) respectively,
containing 10 µM deferoxamine mesylate. PPD derivatives were
prepared in HPLC-grade ACN with 20 mM stock concentration.
Reactions were initiated by adding 3 μL of either PPD, PPD
derivatives, ME-PPD, PTD or control stock solutions for cysteine and
15 μL stock solutions for lysine peptide for a total incubation
mixture volume of 300 μL. These mixtures were then placed in a
temperature-controlled shaking incubator at 25°C.28,29 At each time
point of cysteine and lysine peptides (24 h), the incubation mixtures
were quenched with 300 µL of 95% ACN in water mixture
containing 3 μg/mL of internal standard leucine encephalin. For
cysteine DPRA, 10 μL of each sample mixture was diluted in 380 μL
of 2% ACN in water. Then 10 μL of 16 mM 1,4-dithiothreitol solution
Synthesis of unsymmetrically aryl capped oligoanilines: 4-Amino-
4′-[(4 -tolylaminomethyl)phenyl]diphenylamine (28). Tin powder
(0.612 g, 5 mmol) was added to a 15 mL conc. HCl suspension of 4-
nitro-4′-[(4-tolylaminomethyl)phenyl]diphenylamine (0.334 g,
1
mmol) and the mixture was refluxed for 18 hours. Upon completion
of the reaction, the colourless solution was diluted with 50 mL of
water and placed in an ice bath and the pH of the mixture was
adjusted to 12. White precipitate appeared was filtered, quickly
washed with water and dried under vacuo. Yield: 0.27 g (88%).
Reddish brown powder of mp 110.6-112.6°C (±0.3-0.4). 1H NMR
(400 MHz, DMSO-d6) δ 7.24 (d, J = 7.6 Hz, 2H), 7.11 (d, J = 7.6 Hz,
2H), 6.78 (s, 1H), 6.68 - 6.64 (m, 5H), 6.47 - 6.45 (m, 5H), 5.58 (s,
1H), 4.48 (s, 2H), 4.14 (d, J = 5.6 Hz, 2H), 2.27 (s, 3H). 13C NMR (100
MHz, DMSO-d6): δ 142.1, 141.5, 137.6, 135.7, 135.4, 135.3, 128.7,
127.2, 118.6, 118.2, 115.0, 113.4, 47.1, 20.6. HRMS (ESI): calcd for
C20H21N3 [M+H]+: 303.1730, found: 303.1731.
N1-[4-(4-tolylaminomethyl)phenyl]-N4-(4-aminophenyl)-1,4-
benzenediamine (29). To the dark red solution of nitro compound (freshly prepared in Milli-Q water) was added to prevent
(0.505 g, 1.2 mmol) in 20 mL ethanol, 1.5 g (7.91 mmol) stannous dimerization of the thiol groups and degradation of di-adducts to
chloride was added followed by 5 mL conc. HCl. Resulting mixture mono-adducts.26 Thereafter, the samples were incubated at 45°C
was refluxed for 18 hours. After cooling the reaction mixture was for 30 min, then allowed to cool to room temperature prior to
diluted with water and pH of the solution was adjusted to 10 by analysis LC-MS/MS. For lysine DPRA, DTT solution is unnecessary
addition of 20% aqueous NaOH solution. The cloudy mixture was and 10 µL of above quenched mixture was diluted into 390 µL 2%
extracted with ethyl acetate and organic layer was washed with ACN in water mixture before the analysis. A standard calibration
water, brine and dried over anhydrous sodium sulphate and curve was prepared for the cysteine and the lysine peptides.
evaporation of solvent afforded the title compound as dark green Cysteine peptide standards was prepared in a solution of 20%
powder. Yield: 0.34 g (72%).: dark green powder of mp 143.5-145.5 ACN:buffer using phosphate buffer (pH 7.5) for the cysteine peptide
1
°C (±0.1-0.3). H NMR (400 MHz, DMSO-d6) δ 7.26 (d, J = 8.0 Hz, and ammonium acetate buffer (pH 10.2) for the lysine peptide.
This journal is © The Royal Society of Chemistry 20xx
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ARTICLE
New Journal of Chemistry
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Analysis was performed to detect cysteine, lysine and internal This work was supported by the National University of
View Article Online
DOI: 10.1039/C9NJ03362A
standard leucine enkephalin selectively. The MRM of cysteine (m/z Singapore (NUS), Department of Pharmacy (AcRF Tier 1 FRC
750.3-120.1), lysine (m/z 389.0-129.30) and leucine encephalin (m/z grant R-148-000-267-114), A-STAR SERC (grant number: 152 80
555.6-120.2) were optimized to detect analytes and IS with high 00046 (R-148-000-222-305)), and R-148-000-227-720.
selectivity and sensitivity.
8
9
Hair dyeing
Notes and references
Dye preparation. Hair dye formulations were prepared from PPD
1. K.S. Mukkanna, N.M. Stone and J.R. Ingram, J. Asthma.
and the derivatives PPD 1, 6, 7, 8, 10, PTD and ME-PPD. Three types
of formulations were prepared, following the methods of Shah et al
10
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derivatives with or without using hydrogen peroxide. Formulation A
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Mortz, D. Orton, M. Worm A. Muraro, P. Schmid-Grendelmeier,
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5.https://www.contactderm.org/i4a/pages/index.cfm?pageid=3
is an aqueous solution prepared without an oxidizing agent.
Formulation B contains 3% H2O2. Formulation C contains the
coupler resorcinol and 3% H2O2. Derivatives were dissolved in
deionized water and the pH of the dye was then adjusted to pH 9.5-
10 using 20% aqueous Na2CO3 solution, before the addition of H2O2.
Hair samples approximately 35 cm in length and weighing 2-5 g
were used for dyeing. The dye formulations were applied to
467, 12th November 2018.
swatches of naturally black Asian hair samples bleached for 20 min.
6. W. Meuling, F. Hueber-Becker, L. Roza, F. Benech-Kieffer, J.
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dried for 20 min, and washed with deionized water to remove any
excess dye. They were then washed twice with Silkpro VitAir Series
daily balance shampoo, further treated with Silkpro VitAir Series
daily treatment Masque (hair conditioner) for 5 min and air dried.
Hair colors were measured 1 day later as well as once a week
following washing for 4 weeks and then continued every month for
6 months.
10. G.F. Gerberick, J.A. Troutman, L.M. Foertsch, J.D. Vassallo,
Hair color measurement. The SkinColorCatch (Delfin Technologies
M. Quijano, R.L.M. Dobson, C. Goebel and J.P. Lepoittevin,
Ltd, Kuopio, Finland) was used to measure the CIELAB tristimulus
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L*, a* and b* values of the dyed hair samples. L* indicates
11. D. Jenkins and E.T. Chow. Australas. J. Dermatol. 2015, 56,
brightness from black (L* = 0) to white (L* = 100), a* is the green
40.
(negative, to -128) to red scale (positive, to +127) and b* the blue
12. (a) M. Kock, P. J. Coenraads, B. Blömeke and C. Goebel, Br. J.
(negative, to -128) to yellow scale (positive, to +127). L0, a0 and b0
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are baseline values measured from natural, untreated hair. The
Coenraads, J. Hennen, M.Kock and C.Goebel, Br. J. Dermatol,
휟푯
difference in the color tone is given by
2015, 172, 976. 9.
(a * - a0)2 + (b * - b0)2
횫푬 =
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and the total color difference by
(L * - L0)2 + (a * - a0)2 + (b * - b0)2
.
Conflicts of interest
The hair dyes included in this article are covered by a patent
(PCT/SG2018/050571) filed by the National university of
Singapore in November 2018.
Acknowledgements
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Chemical modification of the hair dye para-phenylenediamine
results in colors that avoid in situ oxidation and toxicity