Phototoxicity of 7-oxycoumarins with keratinocytes in culture

Article abstract of DOI:10.1016/j.bioorg.2019.103014

Seventy-one 7-oxycoumarins, 66 synthesized and 5 commercially sourced, were tested for their ability to inhibit growth in murine PAM212 keratinocytes. Forty-nine compounds from the library demonstrated light-induced lethality. None was toxic in the absence of UVA light. Structure-activity correlations indicate that the ability of the compounds to inhibit cell growth was dependent not only on their physiochemical characteristics, but also on their ability to absorb UVA light. Relative lipophilicity was an important factor as was electron density in the pyrone ring. Coumarins with electron withdrawing moieties – cyano and fluoro at C₃ – were considerably less active while those with bromines or iodine at that location displayed enhanced activity. Coumarins that were found to inhibit keratinocyte growth were also tested for photo-induced DNA plasmid nicking. A concentration-dependent alteration in migration on neutral gels caused by nicking was observed.

Full text of DOI:10.1016/j.bioorg.2019.103014

BioorganicChemistry89(2019)103014
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Phototoxicity of 7-oxycoumarins with keratinocytes in culture
⁎
Christophe Guillona, , Yi-Hua Janb, Diane E. Heckc, Thomas M. Marianob, Robert D. Rappa,
Michele Jettera, Keith Kardosa, Marilyn Whittemored, Eric Akyeaa, Ivan Jabine, Jeffrey D. Laskinb,
Ned D. Heindela
a Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA
b Department of Environmental and Occupational Health, Rutgers University School of Public Health, Piscataway, NJ 08854, USA
c Department of Environmental Science, New York Medical College, Valhalla, NY 10595, USA
d Buckman Laboratories, 1256 N. McLean Blvd, Memphis, TN 38108, USA
e Laboratoire de Chimie Organique, Université Libre de Bruxelles, B-1050 Brussels, Belgium
A R T I C L E I N F O
A B S T R A C T
Keywords:
Seventy-one 7-oxycoumarins, 66 synthesized and 5 commercially sourced, were tested for their ability to inhibit
growth in murine PAM212 keratinocytes. Forty-nine compounds from the library demonstrated light-induced
lethality. None was toxic in the absence of UVA light. Structure-activity correlations indicate that the ability of
the compounds to inhibit cell growth was dependent not only on their physiochemical characteristics, but also
on their ability to absorb UVA light. Relative lipophilicity was an important factor as was electron density in the
pyrone ring. Coumarins with electron withdrawing moieties – cyano and fluoro at C3 – were considerably less
active while those with bromines or iodine at that location displayed enhanced activity. Coumarins that were
found to inhibit keratinocyte growth were also tested for photo-induced DNA plasmid nicking. A concentration-
dependent alteration in migration on neutral gels caused by nicking was observed.
7-hydroxycoumarins
7-oxycoumarins
Furocoumarins
Psoralens
Methoxsalen
8-MOP
Phototoxicity
PAM212 keratinocytes
DNA photo-damage
1. Introduction
modest phototoxicity (IC50 476 μM) for umbelliferone (7-hydro-
xycoumarin) against the MCF-7 breast carcinoma cell line [11]. In ad-
Simple monomeric 7-hydroxycoumarins (or their glycosides) as well
as linear fused-ring furocoumarins, a.k.a, psoralens, are widely dis-
tributed in many dicotyledonous plant families, including the Apiaceae
(Umbellifereae), Asteraceae, Fabiaceae, Moraceae, Rosaceae, Rubiaceae,
Rutaceae and Solanaceae as well as in monocotyledonous plants, such as
Gramineae, palms, lilies, and orchids [1]. In traditional and in estab-
lished medicine, these compounds have found broad pharmaceutical
utility as anti-microbials, immune-modulators, anti-inflammatories,
therapeutics for hyperproliferative skin diseases, anti-virals, and cancer
chemotherapeutics [2,3].
dition, Goard has shown that a conjugate containing 6-bromo-7-hy-
droxycoumarin, attached at C4 to the antibiotic anisomycin, served as a
photo-activated inhibitor of protein biosynthesis better than did the
parent antibiotic itself against CHO or HEK293 cells [12]. Indeed, some
coumarins can display phototoxic effects while others can display a
structure-specific avidity for DNA binding although not by intercala-
tion. Coumarin, esculetin, and a set of pyridyl-substituted coumarins
formed reversible complexes with DNA; physical measurements sup-
ported their binding loci to be in the minor groove by a weak hydrogen-
bonding association of the DNA with the coumarin carbonyl [13–15].
Most psoralens, however, intercalate DNA and photo-crosslink at the
pyrimidine bases [16]. Thus, coumarins and psoralens exert their
pharmacology by different mechanisms.
8-Methoxypsoralen, a.k.a., methoxsalen or 8-MOP, and many si-
milar furocoumarins, have an especially long and extensive literature as
phototoxin/phototherapeutics [4–6]. However, the story on photo-
toxicity of the 7-hydroxycoumarins is more limited and ambiguous.
Several researchers have concluded that “simple coumarins (umbelli-
ferone), show no phototoxic effects” [4,7–9]. But, nevertheless, a
number of coumarins – substituted with electron donating groups –
were reported to be photo-labile and undergoing a 2 + 2 cycloaddition
(dimerization) at the C3-C4 double bond [10]. Mousavi demonstrated a
The psoralens and the 7-hydroxycoumarins are related in several
ways. Virtually all the laboratory synthetic pathways to the linear
furocoumarins pass through the functionalized 7-hydroxycoumarins as
the penultimate precursors of the resulting three-ring psoralens [16].
Nature also uses these same 7-hydroxycoumarins on the bio-synthetic
pathway to the natural product psoralens [16,17]. Besides sharing
⁎ Corresponding author.
E-mail address: chg3@lehigh.edu (C. Guillon).
https://doi.org/10.1016/j.bioorg.2019.103014
Received 8 February 2019; Received in revised form 23 May 2019; Accepted 24 May 2019
Availableonline25May2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

C. Guillon, et al.
BioorganicChemistry89(2019)103014
structural features and a similar synthetic pathway, the psoralens and
the 7-hydroxycoumarins also share comparable absorption and quali-
tatively similar fluorescence behaviors. Methoxsalen. which absorbs at
220, 250, and 305 nM, can be excited at 250 nm, and thereafter gives a
weak fluorescence emission at 505 nM with a quantum yield of 0.002
[18]. Similarly, 7-hydroxycoumarin also has broad absorption bands at
220, 255, and 295 with a λmax of 330 nm and weak shoulder end-ab-
sorption at 370 nm. However, when excited at 330 nm it emits such
intense blue fluorescence at 455 nm (with an impressive quantum yield
of 0.81) that the compound finds wide use as a fluorescent whitener in
laundry detergents [19–21]. Because of their acidic phenolic eOH, 7-
hydroxycoumarins show a range of pH-dependent excitations and
emissions with quantum yields approaching unity above pH 9.4 [21].
The psoralens show no such pH-dependency.
in preclinical development.
2. Results and discussion
While coumarins including the 7-hydroxy compounds, their ethers
and alkylated analogs, have shown broad promise as anti-in-
flammatories, anti-tumor, anti-HIV, anticoagulant, antimicrobial, anti-
oxidant, hepatoprotective, anti-pyretic, anti-hypertensive, and anti-
convulsant agents, they nevertheless display complex pharmacologies,
which are truly difficult to correlate [27]. The problem is that for any
given compound library, no validated quantitative structure-linked
comparative principle has yet to be found although logP, electronic
surface analysis, pKa, and size have all been considered. In coumarin
papers, structure-activity relations are phrased in general terms and
even then, any given set displays many exceptions. For example, “for
TNF-α inhibitory effects…the C6 position must contain a halogen or
methoxy…hydrophobic groups at C3 are favored…small aliphatic
groups at C4 are optimal.” [27]. Similar general statements are reported
for anti-HIV coumarins [28] and for anti-fungal coumarins [29]. For
instance, Kostova noted the anti-HIV activities for substituents on C4,
were ”methyl > H > propyl > benzyl” but he similarly noted that
“to describe structure-activity relationships on the potent anti-HIV
coumarin derivatives is not an easy task.” The anti-fungal SARs for a
series of 24 diverse coumarins were equally problematic. Guerra re-
ported that all ethers of 6-hydroxycoumarin were inactive in his anti-
fungal assay (the parent phenol did have modest activity) but the ethers
of 7-hydroxycoumarin were active and with declining effect as the 7-
alkoxy chain lengthened [29]. No other correlations were made.
In the present studies, PAM212 mouse keratinocytes were used to
evaluate the biological activity of the coumarins [30]. In this assay the
PAM212 cells were treated with the coumarins and then exposed to
UVA light. Control cells were treated with the coumarin without UVA
light. The cells were then allowed to grow for 4–5 days and then ana-
lyzed for growth. Fig. 1 shows typical keratinocyte growth inhibition
experiments with several of the coumarins. Structure activity analysis
for the photobiological activity of the coumarins is shown in Table 1.
Without UVA light treatment, no significant growth inhibition by the
coumarins is evident. However, following UVA light treatment, there is
a marked inhibition of cell growth. Table 1 summarizes the con-
centration of the coumarins inhibiting growth by 50% (IC50) for the
Because the phenolic-capped O-acylated and the O-glycosylated 7-
hydroxycoumarins – like the corresponding psoralens – display almost
no fluorescence, they can serve as useful fluorogenic substrates for
quantifying enzymatic activity (hydrolytic and glycolytic) [22,23].
Thus, enzymatic liberation of the highly fluorescent free phenol pro-
vides a readily measured marker; molecular orbital calculations have
been advanced to explain this phenomenon [24].
Intrigued by the structural commonalities, the spectral dissim-
ilarities and the ambiguous reports on the photobiology of umbellifer-
ones,
a study of the phototoxic effects of seventy-one 7-hydro-
xycoumarins, their ethers and esters, (see Fig. 1 and Table 1) was
undertaken. It is well recognized that ultraviolet light irradiation in
combination with chemical photosensitizers, such as psoralens, has
considerable biological activity in the skin. In addition to causing er-
ythema, sunburn, and tanning, the photoactivated psoralens cause al-
terations in keratinocyte growth and differentiation [25]. Indeed, be-
cause of these biological responses, the psoralens have been used to
treat many skin disorders, particularly, eczema and psoriasis [26].
Many derivatives of psoralens have been evaluated in phototoxicity
assays using keratinocyte growth inhibition to evaluate biological ac-
tivity. In the present studies we used a mouse keratinocyte cell line to
evaluate phototoxicity of a number of structural analogs of the 7-oxy-
coumarins. PAM212 cells in culture are a common mouse keratinocyte
line often used to investigate mechanisms of drug action since results
with these cells can lead directly to in vivo mouse models and subse-
quently to human keratinocytes as lead pharmaceuticals are advanced
Fig. 1. Effects of coumarins on keratinocyte growth with and without UVA light. PAM212 cells were grown in tissue culture dishes and treated with increasing
concentrations of the coumarins. After 30 min at 37 °C, cells were treated with (closed symbols) or without (open symbols) UVA light. Cells were then referred with
fresh growth medium. After 4–5 days, cells were removed from the dishes and counted on a Coulter Counter. Data are presented as percent inhibition of cell growth
relative to controls. Data represent means
SEM (n = 3). In a typical experiment, control plates contained 5.5 × 104 cells.
2

C. Guillon, et al.
BioorganicChemistry89(2019)103014
Table 1
Effects of 7-Oxycoumarins plus UVA light on the growth of PAM212 keratinocytes.
Cpd
Source
Melting point (°C)
R1
R2
R3
R4
R5
IC50 (μM)
1
A
S
S
S
S
S
S
S
S
S
S
B
S
S
S
S
S
S
S
S
S
S
S
S
C
S
S
S
S
S
D
C
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
228–230a
H
H
H
eOH
H
> 100
4
2
145–146
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
eOC(Me)2C^CH
eOCH2CH]CMe2
eO(CH2)5Me
eO(CH2)3Me
eO(CH2)2Me
eOCH2CH]CHMe
-OMe
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
3
93–94
H
H
10
4
83–84
H
H
10
5
106–108
H
H
10
6
103–104
H
H
15
7
114–116
H
H
33
8
166–167 [31]
139–141 [32]
87–88.5
H
H
40
9
H
H
eOCH2C^CH
eOCH2C^C(CH2)4Me
eO(CH2)7Me
eOH
41
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
H
H
60
80–82
H
H
60
260.5–262
158–159.5 [33]
146–147
H
H
100
1
H
I
eOCH2CH]CH2
eOCH2CH]C(Me)2
eO(CH2)2Me
eOH
H
I
5
124.5–126
219–220 [33]
145.5–147.5
229.5–231.5
199–200
H
I
10
H
I
18
H
NH2
eOCH2CH]CH2
eOH
5
H
NO2
> 100
12
eCH2CH]C(Me)2
H
eOH
165–167
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
eCH2CH]C(Me)2
eOH
1
168–170 [34]
175–176
eCH2CH]CH2
eOH
10
eCH(Me)CH]CH2
eOH
14
158–160
eCH2CH]CHMe
eOH
20
149.5–150 [35]
159–161
eCH2CH]CH2
-OAc
21
H
-OMe
60
101–102 [36]
254–256 [32]
151.5–153
129–130
H
eOCH2CH]CH2
eOH
H
10
H
I
> 100
22
H
eOCH2eCH]C(CH3)2
eO(CH2)2Me
-OMe
I
H
I
50
194–195
H
I
70
119–120
H
-OMe
OMe
H
33
118–121
H
H
-OMe
50
182–183
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
Me
Me
Me
Me
H
eCH2CCl]CH2
eOH
Me
Me
Me
H
20
143–144.5
112–113
H
eOCH2(CO)Me
-O(CH2)2Me
eOCH2CH]CH2
eOH
40
H
100
> 200
> 200
> 300
> 100
> 100
> 100
> 100
> 300
> 300
> 300
> 300
> 300
21
76–77
H
197–198
H
Me
Me
H
160.5–161.5
97–98
H
eOCH2C^CH
eOCH2CH2CH3
-OAc
H
151–152
eCH2CH]CH2
Me
H
112.5–113.5 [37]
110.5–112
174–175
H
-OMe
H
eOCH2C^CH
eOH
H
eCH2CH]CH2
Me
Me
Me
H
114.5–115.5
109.5–111 [38]
158–159
H
eOCH2CH]CH2
-OMe
H
H
eOCH2(CO)Me
eOH
186–188 [39]
149.5–150 [35]
198–198.5(d) [40,41]
158–160 [40,41]
269–271 [42]
143–143.5
131.5–133
139.5–140
181–182
H
H
eCH2CH]CH2
-OAc
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
eCH2(CHBr)CH2Br
eOH
1
eCH2CH]CH2
eOH
5
H
H
H
H
H
H
H
H
eOH
10
eOCH2CH]CH2
eOCH2CH]CMe2
OCH2CH2CH3
eOCH2C^CH
-OMe
2
H
5
H
5
H
10
154–155
H
13
209–211 [42]
147–148
H
eOH
20
Me
eOCH2CH]CH2
5
(continued on next page)
3

C. Guillon, et al.
BioorganicChemistry89(2019)103014
Table 1 (continued)
Cpd
Source
S
Melting point (°C)
231–232
R1
Br
R2
R3
R4
R5
IC50 (μM)
20
59
Me
-OAc
Me
60
61
62
63
64
65
66
67
68
69
70
71
S
S
S
S
S
S
S
S
S
S
S
S
158–160 [41]
Br
Br
CN
CN
CN
CN
CN
F
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
eCH2(CHBr)CH2Br
eCH2CH]CH2
eCH2(CHBr)CH2Br
eCH2(CHBr)CH2Br
eCH2CH]CH2
eCH2CH]CH2
H
-OAc
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
48
160–161 [41]
-OAc
140
221–223 [40,41]
170–171 [41]
eOH
3
-OAc
48
234–235 [40,41]
120–124(d) [40,41]
172–173 [40,41]
175–176 [40,41]
192–193 [43]
eOH
> 300
> 300
> 300
120
-OAc
eOCH2CH]CH2
eOH
eCH2(CHBr)CH2Br
H
F
eOH
> 200
> 300
> 300
61
117–118 [40,41]
163–164 [40,41]
228–230(d)
F
H
eOCH2CH]CH2
eOH
F
eCH2CH]CH2
H
I
eOH
a
Compound 1 is umbelliferone also known as 7-hydroxycoumarin. An IC50 expressed as > (number) means no activity was observed up to that value, usually the
solubility limit of the compound for the assay’s conditions. A = Sigma-Aldrich; B = Regis Chemical; C = TCI America Inc.; D = Elder (Valeant) Pharmaceuticals. S
means that the compound was synthesized for these studies either following an existing procedure or a new one developed for that purpose, which is described in
detail in the experimental part. Published methods for specific coumarins are indicated by references provided in the table and in the experimental part. Obvious
(d)
decomposition upon melting is noted by
.
compounds in Fig. 1 as well as other coumarins in the data set com-
pounds (1–71).
physiological pH and in this case with clogP = 2.94 displays an IC50 of
5 μM.
Based on these data (Table 1), lipophilicity is an important factor.
Compare for example the parent 7-hydroxycoumarins and their corre-
sponding 7-alkoxy ethers. Dimethylumbelliferone, (12) logP = 2.1
displayed a modest IC50 of 100 μM while all its ethers (2–11) displayed
substantially improved activity. The lower chain length ethers, com-
pounds 2–9, (whose cLogP values fell between 3.2 and 3.9 were con-
siderably more active than were the longer chain length ethers (10 and
11) for which cLogPs of > 5.8 reflected an enhanced – and apparently
less optimal – lipophilicity.
In the 8-iodo series (27–30) the parent phenol, 4-methyl-8-iodo-7-
hydroxycoumarin (27, cLogP = 2.93) was inactive while its more li-
pophilic 7-alkoxy variants (28, 29, and 30 with cLogPs of 4.81, 4.16,
and 3.10 respectively) had IC50′s of 22, 50, and 70 μM respectively.
Here the activity of the alkoxy compounds does increase with the in-
creasing lipophilicity as provided by the carbon chain but since no
analog has more than a five-carbon side chain a maximum lipophilicity
on the Hansch curve has not been attained.
In keeping with the trend that the alkoxys are more photo-active
than their parent phenols, as noted umbelliferone (7-hydro-
xycoumarin = 1) showed no activity in the screen; but its O-methyl
ether (7-methoxycoumarin = 32) had an IC50 of 50 μM while its bis-
methyl ether (7,8-dimethoxycoumarin = 31) had an IC50 of 33 μM.
The two families of fluoro-substituted coumarins constitute diverse
classes. The first of these, those bearing a trifluoromethyl at R2, position
4 in traditional coumarin numbering, (e.g., 33–47), were largely in-
active. The two parent phenols, compound 37 (4-trifluoromethyl-8-
methyl-7-hydroxycoumarin) and the nor-methyl compound 47 (4-tri-
fluoromethyl-7-hydroxycoumarin), were inactive at their solubility
maxima. The traditional alkyl, alkenyl, or alkynyl ethers of 47 (e.g., 36,
39, 41, 42, and 46) which in the other 7-hydroxy structural families did
impart significant activity over their less active parent phenols, were
equally inactive in this particular 4-trifluoromethyl family (the one
with no 8-methyl moiety). However, three analogs of phenol 37 (the 4-
trifluoromethyl-8-methyl-7-hydroxy system), were active (viz., 33–35).
Is this observed activity an effect of increased lipophilicity or is it de-
rived from an electronic effect? The answer is probably both since two
of the active derivatives (33 with cLogP = 4.09 and 35 with
cLogP = 4.20) were indeed much more lipophilic than 37
Furthermore, when the alkenyl or alkyl moiety was rearranged from
the 7-position (where it was an ether) onto either ring positions 3- or 6-
(e.g., 19–24), all of these 7-hydroxycoumarins were more active (IC50′s
1–21 μM) than the parent dimethylumbelliferone (12) from which they
were synthetically derived. In addition, all these 3- or 6- alkylated 7-
hydroxycoumarins were more lipophilic than 12 with their cLogPs
falling between 3.6 and 4.6.
Similarly, in the 6-iodo series, the parent phenol, 6-iododimethy-
lumbelliferone (16 with clogP = 3.38) possessed an IC50 of 18 μM –
much more photo-active than its nor-iodo, the dimethylumbelliferone
(12, logP = 2.10) – but even here its more lipophilic ethers the 6-iodo-
7-alkoxy derivatives (viz., 13–15) with clogPs of 4.3–5.4, were all more
active. The 4,8-dimethyl-6-nitro-7-hydroxycoumarin (18) is a member
of the family of acidic ortho-nitrophenols (in general pKa’s average 7.2
for o-nitrophenols and clogPs average 1.7), which are extensively io-
nized at physiological pH and thus might be expected to display poor
membrane permeability. At a cLogP of 2.39 compound 18 possesses no
photocytotoxicity up to its solubility maximum of 100 μM. On the other
hand, the 4,8-dimethyl-6-amino-7-allyloxycoumarin (17), a member of
the family of weakly basic aromatic ortho amino ethers, is unionized at
4

C. Guillon, et al.
BioorganicChemistry89(2019)103014
Fig. 2. Ability of coumarins and UVA light to
damage plasmid DNA. Plasmid DNA was treated
with increasing concentrations of the coumarins.
Lane L shows products from the digestion of the
plasmid with restriction endonuclease EcoRI to
linearize the DNA (100 ng); in native gels, the
linearized DNA migrates as one double-stranded
band, whereas, in denaturing gels, it migrates as
two well-separated single strands. Lane S shows
untreated plasmid DNA (75 ng); in native gels,
the intact plasmid DNA migrates mainly as one
supercoiled double-stranded band, whereas in
denaturing gels, it migrates as two closely mi-
grating single strands. Plasmid was analyzed in
neutral (panels A and B) or alkaline (panels C
and D) agarose gels. Plasmid samples were
treated with (panels B and D) or without (panels
A and C) UVA light.
(cLogP = 3.00), the inactive parent phenol, but the 7-O-acetonyl analog
(34) displayed equivalent lipophilicity (clogP = 2.50) to the parent and
yet a substantially enhanced 40 μM activity. There may be an ex-
planation for the activity (IC50 = 20 μM) seen in 33 with its pendant
chloro-containing side chain. Platz has claimed an augmented photo-
induced anti-virual activity for sensitizable heterocyclics with halogen-
bearing side chains [44]).
10 μM or less. The heavy-halogen class of coumarins is the most photo-
active. The significant phototoxic activity observed in the 3-brominated
/iodinated coumarins (viz. 49–61 and 71) as well as in those derived
from dibrominating the C6 (R3) allyl (viz. 49, 60, 62, 63, and 67), may
arise from the well-known photo-homolytic cleavage of these labile
carbon-halogen bonds [46]. Not only is there a synthetically useful
photo-radical cleavage which uses the 3-brominated/iodinated cou-
marins as starting materials for a coupling reaction [47] but also such 3-
bromo/iodo species have demonstrated potent photo-viricidal proper-
ties [48]. Park and colleagues advanced a theory in which 3-bro-
moumbelliferone – with a 7-alkoxy side chain to provide affinity to a
viral nucleic acid strand – was photolyzed to a free radical at C3 which
subsequently effected a cleavage of that strand [48]. While other non-
fluoro halogens (Cl, Br, and I) attached at C3 on the coumarin ring were
claimed efficacious as photo-activated radical producers [48], Platz also
noted that halogens on an aliphatic side chain off the coumarin ring
were equally efficacious as light activated anti-viral agents [44]. In the
set of coumarins (see 49, 60, 62, 63 and 67), it was demonstrated that
the 2,3-dibromopropyl side chain substantially augmented the photo-
toxicity of these dibromoalkyl compounds over that of close structural
analogs lacking that moiety.
The second family of fluoro-substituted coumarins, compounds 67
to 70 bearing an F at R1, position 3 in the traditional coumarin num-
bering, was similarly an inactive class. Not only the parent phenol (3-
fluoro-4,8-dimethyl-7-hydroxycoumarin, 68) but also the O-allyl and C-
allyl analogs displayed no activity up to their solubility maxima. One
exception, compound 67, with a 2,3-dibromopropyl sidechain, was
modestly active at 120 μM probably by a free radical effect to be dis-
cussed later.
We are not the first to observed that fluoro-attachment can be a
deactivating structural feature in otherwise promising umbelliferones
and other coumarins as candidate therapeutics. A CF3 attachment at
ring positions 3 or 4 (R1 and R2 in the generalized coumarin structure,
see Figure on Table 1) was found to abolish otherwise significant anti-
inflammatory activity in a study of more than 50 diverse coumarins
[27]. In addition, fluorine attachment to the ring carbons of 7-hydro-
xycoumarins has been found to impart a photostability, an inertness to
photo-reactivity and a resistance to fluorescence quenching; these
properties all being problematic to the successful use of non-fluorinated
umbelliferones as fluorophors in biological systems [22]. The photo-
inactivity of the two types of fluoro coumarins in this study is therefore
to be expected.
In summary, we established structural trends within the set of 71
coumarin derivatives presented herein. None of the compounds dis-
played growth inhibitory activity in the absence of light but 49 of them
did in the presence of UVA light. Lipophilicity played a key role in the
activity of those compounds. Within structural subsets, it was observed
that the more lipophilic derivatives were the most active ones as long as
the lipophilicity of said compounds did not exceed a value beyond
which the activity started to diminish again. The addition of one or
multiple halogens, such as bromo, iodo and chloro, resulted in en-
hanced activity; whether those substitutions occurred at the traditional
ring positions (C3, C6 and C8) or on a more distal side chain. However,
fluorine substitution, in the form of a single fluorine atom or a tri-
fluoromethyl group, typically resulted in partial or complete loss of
activity.
Not surprisingly, the 3-cyano coumarins (e.g., 62–66) show an ac-
tivity profile similar to the trifluoromethyl and fluoro coumarins. The
cyano, the trifluoromethyl, and the fluoro groups are all electronically-
deactivating entities with closely related electron withdrawing sigmas
of 0.56, 0.43, and 0.34 [45]. Very few members of these three electron
withdrawing families display phototoxicity and those that do are the
side-chain brominated analogs. As in the 3-fluoro class (see 67) so in
the 3-cyano class only 62 and 63 (IC50′s 3 and 48 μM), with their 2,3-
dibromopropyl side chains, demonstrated the photosensitization effect.
One unique structure-activity correlation is that all fourteen 3-
bromo- and 3-iodo-coumarins (49–60 and 71) in this study displayed
significant photosensitizing effects with eight of the set having IC50′s of
As psoralens are known to intercalate into DNA, and a few psoralens
are known to bind to the minor groove of DNA [13–15], we next de-
termined if the photo-activated coumarins had the capacity to bind and
damage DNA. For these studies, we used a plasmid DNA nicking and
unwinding assay [30]. Results obtained in this study demonstrate that
5

C. Guillon, et al.
BioorganicChemistry89(2019)103014
Table 2
synthetic methods for all previously unreported coumarins are de-
Percent linearization of plasmid DNA by coumarins plus UVA light.
scribed below.
4,8-Dimethyl-7-[(3-methyl-2-buten-1-yl)oxy]coumarin (3), 4,8-
dimethyl-3-(3-methyl-2-buten-1-yl)-7-hydroxycoumarin (19), and
4,8-dimethyl-6-(3-methyl-2-buten-1-yl)-7-hydroxycoumarin (20)
were all produced in the etherification of 4,8-dimethyl-7-hydro-
xycoumarin (12) when 1.9 g (10.0 mmol) of the latter compound were
dissolved in 49 mL of aqueous sodium hydroxide (containing 0.40 g or
10.0 mmol) and reacted with 1-bromo-3-methyl-2-butene (1.5 g,
10.0 mmol) over 14 hr of vigorous stirring at ambient temperature. The
precipitate which had formed was removed by filtration, washed on the
filter with 20% aqueous sodium hydroxide, and the base-soluble phases
combined. As expected the base insoluble compound (0.44 g, 17%) was
the expected butenyl ether, 4,8-dimethyl-7-[(3-methyl-2-buten-1-yl)
oxy]coumarin (3, mp = 93–94 °C, recrystallized from MeOH).
Elemental analysis and 1H NMR (DMSO‑d6) supported the proposed
structure: δ (ppm) 2.17 and 2.44 (two s, each 3H, ring methyls), 1.68
and 1.83 (two d by second order splitting, each 3H, ](CH3)2), 4.69 (m,
2H, eOCH2e), 5.40 (m, 1H, eCH]), 6.33 (s, 1H, C3eH), 7.43 (d, 1H,
C5-H, J = 8.4 Hz), and 6.90 (d, 1H, C6-H, J = 8.4 Hz).
Concentration
Percentage of nicked plasmid
(µM)
Compd 14 Compd 48 Compd 58 Compd 62 Compd 71
0
0.2
0.2
1.0
1.2
2.2
1
1.0
4.0
3.2
3.2
6.8
10
100
8.0
60.5
90.8
12.0
20.2
55.4
96.6
80.2
98.6
20.5
DNA nicking data from plasmids analyzed in neutral agarose gels (Fig. 2, panel
B). In these experiments, plasmids were treated with increasing concentrations
of the coumarins followed by UVA light. Image analysis was used to calculate
the percent of plasmid unwinding. Note that compounds 48, 62 and 71 were
more effective in nicking DNA.
there is a structure-specific photolytic interaction of coumarins and
DNA. If the candidate coumarin is able to nick and linearize the plasmid
upon light exposure, the change in electrophoretic migration of the
altered DNA is readily evident. (see examples in Fig. 2 and Table 2). A
set of five structurally diverse coumarins was selected from a group that
was found to strongly inhibit keratinocyte growth (see Fig. 1) but which
ranged from halogenated variants, to 7-hydroxyls to 7-allyloxys. There
was little or no damage to the plasmid DNA by any of these compounds
in the absence of UVA. However, with each of the coumarins (14, 48,
58, 62 and 71), UVA light treatment caused concentration-dependent
alteration in migration in neutral gels caused by nicking, which resulted
in a faster migrating supercoiled plasmid when compared to slower
migrating linear or open circular forms of the plasmid. Generally si-
milar results were evident when the plasmid was treated with the
coumarins and UVA light and then analyzed in alkaline gels (14, 48 and
71). These data indicate that the photo-activated coumarins can modify
the DNA plasmid. A direct structure activity correlation between the
phototoxicity results (Table 1) and the plasmid nicking/unwinding re-
sults (Fig. 2 and Table 2) would not be expected, nor was it observed.
The phototoxicity study is a whole cell assay with attendant compli-
cations induced by membrane transport, the presence of growth media,
and metabolism, while photo nicking is performed extracellularly in
simple buffer solution. The C7 hydroxyl molecules 71 and 69 as well as
the pre-hydroxyl (the readily hydrolyzed C7 acetoxy molecule) 48 all
displayed nearly five times greater linearization of the plasmid com-
pared to the 7-allyloxy ethers 14 and 58 although the latter were
among the most phototoxic compounds of the set.
Neutralization with 6 N hydrochloric acid of the aqueous basic
phase precipitated an orange oil which was separated by flash chro-
matography (silica gel, chloroform:ethyl acetate 95:5 elutant) into
three phenols – unreacted starting material (not recovered) and two
different C-alkylated phenols. The first eluted fraction (0.55 g, 21%,
mp = 199–200 °C, recrystallized from MeOH), elemental analysis
within 0.4% of theoretical and 1H NMR (CDCl3) supported the proposed
structure
of
4,8-dimethyl-3-(3-methyl-2-buten-1-yl)-7-hydro-
xycoumarin (19): δ (ppm) 2.32 and 2.36 (two s, each 3H, ring me-
thyls), 1.68 (d, 3H, J = 1.2 Hz, =C(CH3)), 1.78 (s, 3H, =C(CH3)′), 3.35
(d, 2H, J = 6.5 Hz, eCH2e), 5.08 (dd, 1H, J = 6.5 Hz, J′ = 1.2 Hz,
eCH]), 5.86 (br s, 1H, eOH, exchanges with D2O), 6.88 (d, 1H,
J = 8.5 Hz, C6-H), and 7.27 ppm (d, 1H, J = 8.5 Hz, C5-H). The second
component to elute was isolated as off-white short needles (0.31 g,
12%, mp = 165–167 °C, recrystallized from MeOH), elemental analysis
within 0.3% of theoretical and 1H NMR (CDCl3) supported the proposed
structure
of
4,8-dimethyl-6-(3-methyl-2-buten-1-yl)-7-hydro-
xycoumarin (20):
δ
(ppm) 2.36 (s, 3H, C8-CH3), 2.39 (d, 3H,
J = 1.2 Hz, C4-CH3 coupled to C3-H), 1.81 (two overlapping closely
spaced s, 6H, =C(CH3)2), 3.42 (d, 2H, J = 6.8 Hz, eCH2e), 5.30 (m,
1H, J = 6.8 Hz, J′ = 1.2 Hz, eCH]), 5.95 (br s, 1H, phenolic eOH
exchanges with D2O), 6.12 (q, 1H, J = 1.2 Hz, C3-H coupled to C4-CH3),
and 7.18 ppm (s, 1H, C5-H).
4,8-dimethyl-6-allyl-7-hydroxycoumarin (21, mp = 168–170 °C)
and 4,8-dimethyl-6-allyl-7-acetoxycoumarin (24, mp = 149.5–
150 °C) were prepared as reported [34].
3. Experimental
Materials, Methods and Instrumentation: The 7-oxycoumarins
were purchased or synthesized as indicated on Table 1, and in the
following Syntheses. 1H NMR spectra were obtained on a Bruker
500 MHz nuclear magnetic resonance spectrometer in CDCl3 or
DMSO‑d6. All solvents and reagents were of the highest purity com-
Preparation of 4,8-dimethyl-7-[(2-buten-1-yl)oxy]coumarin (7)
and 4,8-dimethyl-6-(2-buten-1-yl)-7-hydroxycoumarin (23). To a
solution of 0.95 g (5.0 mmol) of 4,8-dimethyl-7-hydroxycoumarin
(12), in 30 mL of aqueous sodium hydroxide containing 0.2 g (5 mmol)
of the base, was added 1-chloro-2-butene (0.68 g, 5 mmol). The re-
sulting solution was stirred at room temperature for 18 h, acidified with
10% hydrochloric acid, chilled, and filtered. The dried crude product
was subjected to flash chromatography (silica gel, 99:1 methylene
chloride:2-propanol elutant) with the first product fraction identified as
4,8-dimethyl-7-[(2-buten-1-yl)oxy]coumarin (7, 0.44 g, 36%,
mp = 114–116 °C, recrystallized from methanol/ethyl acetate) and the
second product fraction identified as 4,8-dimethyl-6-(2-buten-1-yl)-7-
hydroxycoumarin (23, 0.47 g, 39%, mp = 158–160 °C). Alternatively,
7 can be prepared without contamination by 23 if the acetone, po-
tassium carbonate phenolic alkylation of 12 is used [49].
mercially available. Combustion analyses were within
0.4 of theo-
retical values (deviations are noted in the text) and NMR spectra (see
below) were in accord with expected resonance shifts. The cLogPs re-
ported herein were obtained from the Chemical Properties calculator in
ChemDraw Professionnal version 17.0. If a measured logP is known,
that value is used in the discussion.
Syntheses: Coumarins from commercial sources or published
syntheses are noted in Table 1 by the name of the supplier or by re-
ference to a published article. The letter S means the indicated cou-
marin was synthesized for this study. In some cases, in which the
published synthesis lacked experimental details or physical properties,
we have repeated that synthesis and the newly measured properties are
reported below. Compounds not present in the text below (8, 9, 31, 32,
50 and 61–70) were prepared according to methods described in the
references displayed in Table 1 for those compounds. In addition, the
4,8-Dimethyl-6-(3-buten-2-yl)-7-hydroxycoumarin (22) was
prepared by the Claisen rearrangement of 0.40 g (16 mmol) of 4,8-di-
methyl-7-[(2-buten-1-yl)oxy]coumarin (7) refluxed for eight hours
under a dry nitrogen stream in 30 mL of N,N-diethylaniline. The cooled
reaction contents were poured into 40 mL of chloroform and extracted
6

C. Guillon, et al.
BioorganicChemistry89(2019)103014
3X with 30 mL each of 25% hydrochloric acid. The organic layer was
washed with 30 mL of water, dried over anhydrous sodium sulfate, and
evaporated in vacuo to a yellow-brown solid, which was crystallized
methyl-2-buten-1-yl)oxy]-8-iodocoumarin (28), isolated as white
microneedles (2.60 g, 55% yield, mp = 151.5–153 °C, recrystallized
from ethyl acetate). Elemental analysis (C,H,I within
0.2% of
from methanol to
a
fluffy beige solid (0.31 g, 78% yield,
theory) and 1H NMR (CDCl3) confirmed the structure: δ (ppm) 1.77 and
1.79 (two s, each 3H, =C(CH3)2), 2.42 (d, 3H, J = 1.2 Hz, C4-CH3),
4.65 (d, 2H, J = 6.8, eOCH2), 5.56 (dt, 1H, J = 6.8, J′= 1.1, eCH]),
6.13 (d, 1H, J = 1.2, C3-H), 6.83 (d, 1H, J = 8.8, C6-H), 7.51 (d, 1H,
J = 8.8, C5-H). Similarly, with n-propyl bromide as the alkylating agent
4-methyl-7-n-propoxy-8-iodocoumarin (29) was obtained (49%
yield, mp = 129–130 °C, recrystallized from ethanol). Elemental ana-
mp = 175–176 °C). Elemental analysis was within
0.1% of theore-
tical and 1H NMR confirmed the structure as 4,8-dimethyl-6-(3-buten-
2-yl)-7-hydroxycoumarin (22). 1H NMR (CDCl3): δ (ppm) 1.41 (d, 3H,
J = 6.8 Hz, C-CH3), 2.32 (s, 3H, C8-CH3), 2.42 (d, 3H, J = 0.98 Hz, C4-
CH3), 3.80 (m, 1H, eCHCH3), 5.15–5.35 (m, 2H, =CH2), 6.12 (q, 1H,
J = 0.98 Hz, C3-H), 7.24 (3, 1H, C5-H).
These 4,8-dimethyl-7-alkoxycoumarins (2, 4, 5, 6, 10 and 11)
were prepared by alkylation of 4,8-dimethyl-7-hydroxycoumarin
(12) (Regis Chemical Company) by the general method for phenolic
etherification [49]. The requisite 7-hydroxycoumarin (1.0 mmol), the
corresponding alkyl/alkenyl/alkynyl bromide (1.5 mmol), 50 mL of
anhydrous acetone, and 3 g of potassium carbonate were stirred vig-
orously at reflux for 16 h, chilled, filtered, and the solid residue washed
on the filter with a minimum of boiling methanol. The organic phase
(acetone, methanol and coumarin ether product) was evaporated and
the crude solid triturated with 20% aqueous sodium hydroxide to re-
move any unreacted 7-hydroxycoumarin. The dried organic solid was
crystallized from methanol to yield the pure ethers in 45–79% yields
with elemental analyses ( 0.4% of theory) and 1H NMR (CDCl3) in
accord with the expected: (2) mp = 145–146 °C, (4) mp 83–84 °C, (5)
mp 106–108 °C, (6) mp 103–104 °C, (10) mp = 87.0–88.5 °C, and (11)
mp = 80–82 °C.
lysis (C,H,I within
0.15% of theoretical) and 1H NMR (CDCl3) con-
firmed the assigned structure: δ (ppm) 1.13 (t, 3H, J = 7.33, eCH3),
1.85 (m, 2H, eCH2e), 2.41 (s, 3H, C4-CH3), 4.10 (t, 2H, J = 6.36,
eOCH2e), 6.14 (s, 1H, C3-H), 6.77 (d, 1H, J = 8.8, C6-H), 7.54 (d, 1H,
J = 8.8, C5-H).
Similarly, by the above method using methyl iodide as the alky-
lating agent, a 54% yield of 4-methyl-7-methoxy-8-iodocoumarin
(30) was obtained (mp = 194–195 °C, recrystallized from ethanol).
Elemental analysis (C,H,I within
0.1% of theoretical) and 1H NMR
(CDCl3) confirming of the assigned structure: δ (ppm) 2.42 (s, 3H, C4-
CH3), 4.00 (s, 3H, eOCH3), 6.15 (s, 1H, C3-H), 6.82 (d, 1H, J = 8.8, C6-
H), 7.57 (d, 1H, J = 8.8, C5-H).
Syntheses of (13–15). 4,8-dimethyl-6-iodo-7-alkoxycoumarins
The allyl, 3-methyl-2-buten-1-yl, and n-propyl ethers were prepared by
the general method of Vyas[49] from condensation of 4,8-dimethyl-6-
iodo-7-hydroxycoumarin (16) and the corresponding bromides (viz.
allyl bromide, 3-methyl-2-butenyl bromide, and n-propyl bromide) in
dry acetone over potassium carbonate in yields of 60–85% (re-
crystallization EtOH). Elemental analyses (C,H) and 1H NMR were in
accord with theoretical values and respective mp’s of the purified ethers
were (13) 158–159.5 °C, (14) 146–147 °C, and (15) 124.5–126 °C.
Syntheses of (52–57): 8-methyl-3-bromo-7-alkoxycoumarins.
The allyl, 3-methyl-2-buten-1-yl, n-propyl, propargyl, and methyl
ethers were prepared by the general method [49]. The parent coumarin,
8-methyl-3-bromo-7-hydroxycoumarin (57), mp = 209–211 °C, was
prepared as described [42] and subsequently condensed with these
corresponding bromides (viz., allyl, 3-methyl-2-butenyl, n-propyl, pro-
pargyl, and methyl bromide) in dry acetone over potassium carbonate
in yields of 68–90%. Elemental analyses (C,H) and 1H NMR were in
accord with theoretical values and the mp’s of the respective purified
ethers were (52) 143–143.5 °C, (53) 131.5–133 °C, (54) 139.5–140 °C,
(55) 181–182 °C, and (56) 154–155 °C.
4,8-Dimethyl-7-allyloxycoumarin (26) was prepared as described
by Kaufman [36].
4,8-Dimethyl-3-bromo-6-[3-(1,3-dioxoisoindolin-2-yl)prop-1-
en-1-yl]-7-acetoxycoumarin (59) was prepared by the heating with
vigorous stirring at 100 °C of 1.85 g (10 mmol) of potassium phthali-
mide in 50 mL of dimethylacetamide with 2.55 g (5 mmol) of 4,8-di-
methyl-3-bromo-6-(2,3-dibromopropyl)-7-acetoxycoumarin (60).
The latter was prepared by our published method [41]. At the end of
five hours the reaction medium was chilled to room temperature in an
ice-water bath and diluted with 300 mL of cold water to precipitate the
organic materials. These were filtered, dried, taken up in methylene
chloride and flash chromatographed. 59 was isolated (0.64 g, 26%
yield, mp = 231–232 °C, recrystallized from CH2Cl2/hexane). 1H NMR
(CDCl3) confirmed the structure: δ (ppm) 2.22 and 2.37 (two s, each
3H, C4-CH3 and C8-CH3), 2.59 (s, 3H, –OAc), 4.44 (dd, 2H,eCH2eN),
6.22 and 6.63 (two m, each 1H, side chain eCH]CHe), 7.53 (s, 1H, C5-
H), 7.73 and 7.85 ppm (two m, each 2H, phthalimido Ar-H).
(58) 4,8-dimethyl-3-bromo-7-allyloxycoumarin was prepared in
52% yield by allyl bromide alkylation in dry acetone/potassium car-
bonate of 4,8-dimethyl-3-bromo-7-hydroxycoumarin (51) by the
general method [49]. The starting material (51) was prepared as re-
ported, mp = 269–271 °C [42]. The title compound (58) appeared from
MeOH as tiny white needles (mp = 147–148 °C). 1H NMR and ele-
mental analysis were in accord with theoretical values.
4,8-dimethyl-7-hydroxy-6-iodocoumarin (16): 4,8-dimethyl-7-
hydroxycoumarin (12, 10.0 g, 52.0 mmol) was dissolved in 80 mL of
dioxane with 200 mL of concentrated aqueous ammonia subsequently
added. A solution prepared from 13.6 g (53.5 mmol) of iodine in
500 mL of aqueous 5% KI was added dropwise with vigorous stirring
over a two-hour period. The greenish color of the solution gradually
disappeared and a yellow precipitate formed. The pH was adjusted to
3.0 with hydrochloric acid, cooled, and filtered to remove the product,
which was taken up in a minimum of ethyl acetate and washed with
water (3 × 50 mL). Evaporation of the ethyl acetate gave 15.7 g (94%)
of the title compound which was recrystallized from MeOH to analy-
tical purity: elemental analysis (C,H), mp = 219–220 °C [33]. 1H NMR
(DMSO‑d6) confirmed that iodination had taken place on C6: δ (ppm)
2.25 and 2.36 (two s, each 3H, CH3′s), 6.13 (s, 1H, C3-H), 7.84 (s, 1H,
C5-H), 10.11 (br S, 1H, OH, exchangeable with D2O).
(37) 4-trifluoromethyl-8-methyl-7-hydroxycoumarin and (47) 4-
trifluoromethyl-7-hydroxycoumarin, were prepared as described
[50] and had melting points (197–198 °C and 186–188 °C respectively)
and 1H NMRs in accord with those expected.
Synthesis of (34, 35, 38, 44, and 45), 4-trifluoromethyl-8-me-
thyl-7-alkoxycoumarins. The 2-ketopropyl, n-propyl, propargyl, allyl,
and methyl ethers were prepared by the general method [49] from
condensation of 4-trifluoromethyl-8-methyl-7-hydroxycoumarin
(37) with chloroacetone, n-propyl bromide, propargyl bromide, allyl
bromide and methyl iodide in yields of 31 to 56% (recrystallization
from EtOH). Elemental analyses (C,H) and 1H NMR were in accord with
theoretical values and respective mp’s of the purified ethers were (34)
143–144.5 °C, (35) 112–113 °C, (38) 160.5–161.5 °C, (44)
114.5–115.5 °C, and (45) 109.5–111 °C.
4-Methyl-8-iodo-7-hydroxycoumarin (27) was prepared as de-
scribed in 35% yield, mp as reported 254–256 °C [32].
4-Methyl-7-alkoxy-8-iodocoumarins, (28–30). A solution of 27
(3.87 g, 12.8 mmol), 2 g of 1-chloro-3-methyl-2-butene (19.1 mmol),
8.0 g potassium carbonate (58 mmol), and 200 mL of acetone was
stirred at reflux for four hrs. After filtration the potassium carbonate
was washed on the filter three times with hot acetone. Combined fil-
trates and washings were evaporated and the product, 4-methyl-7-[(3-
Synthesis of (36, 39, 41, 42, and 46). These 4-trifluoromethyl-7-
alkoxycoumarins were prepared by etherification of 4-tri-
fluoromethyl-7-hydroxycoumarin (47) with allyl bromide, n-propyl
7

C. Guillon, et al.
BioorganicChemistry89(2019)103014
bromide, methyl iodide, propargyl bromide, and chloroacetone in 28 to
58% yields (recrystallized from EtOH) with elemental analyses (C,H
(ppm) 9.43, 18.14, 74.57, 113.64, 115.13, 116.00, 118.69, 120.09,
125.52, 133.48, 147.0, 151.49, 152.50, 159.62.
within
0.3% of theory) and 1H NMR in accord with theory. The
(71) 3-Iodo-4,8-dimethyl-7-hydroxycoumarin. Finely pulverized
4,8-dimethyl-7-hydroxycoumarin (12) (3.00 g, 15.7 mmol) was
added to 150 mL concentrated acetic acid with subsequent dropwise
addition of ICl (5.35 g, 33.0 mmol). This mixture was heated at 50 °C
with stirring overnight. The mixture was cooled and the crystals col-
lected by filtration. The yield was 3.22 g (65%). The white crystals were
purified by recrystallization from methanol, with mp = 228–230 °C.
C,H,N analysis was within + 0.10% of theoretical. 1H NMR (DMSO‑d6):
δ (ppm) 2.14 and 2.58 (two s, each 3H, C4-CH3 and C8-CH3), 6.84 (d,
1H, C6-H, J = 8.7 Hz), 7.55 (d, 1H, C5-H, J = 8.7 Hz), 10.53 (br s, 1H,
eOH).
respective mp’s of the purified ethers were (36) 76–77 °C, (39)
97–98 °C, (41) 112.5–113.5 °C, (42) 110.5–112 °C, and (46)
158–159 °C.
Syntheses of (33, 43 and 40). These 6-allyl-substituted coumarins
were prepared by the Claisen rearrangement of the corresponding 7-
hydroxy ethers. The rearrangement procedure in N,N-diethylaniline as
described above for preparation of compound (22) was followed. First,
4-trifluoromethyl-8-methyl-7-hydroxycoumarin (37) was etherified
by Vyas’s method [49] with 2-chloroallyl chloride (also known as 2,3-
dichloropropene) and with allyl chloride respectively. The crude ethers
were evaporated to dryness from the acetone solvent used to prepare
them and each was solubilized in N,N-diethylaniline (five times the
weight of aniline per unit weight of allyl ether) and heated at reflux
under nitrogen for 12 h. Work-up and product isolation as described for
(22) gave 24% yield of chloroallyl (33), light tan needles, mp
4. Biological evaluation – experimental methods
Growth inhibition assays: Assays to measure keratinocyte growth
inhibition were performed as previously described [30]. Briefly,
PAM212 mouse keratinocytes (25,000 cells per well) were inoculated
into 6-well Falcon plastic tissue culture dishes in Dulbecco's Modified
Eagle's medium supplemented with 10% newborn calf serum in an in-
cubator containing 5% carbon dioxide at 37 °C. After 24 h, the medium
was changed to fresh growth medium supplemented with control
medium or medium containing increasing concentrations of the cou-
marins. The medium contained 10% serum and 0.2% DMSO which
facilitated the solubilization of the coumarins. After an additional
30 min, the uncovered culture plates were exposed to UVA light
(320–400 nm) emitted from a bank of four BLB fluorescence light tubes
(F40 BL, Sylvania) placed approximately 10 cm above the cell culture
plates. In these experiments, the incident light on the culture plates was
2.4 mW/cm2 as measured with an International Light UV radiometer,
Model IL442A. The cells were exposed to 1.28 J/cm2 of UVA. After
exposing the cells to UVA light, the cell culture medium was drained
and the cells refed with fresh growth medium. After incubating the cells
for 4–5 days, cells on the plates were removed with trypsin and counted
with a Coulter Counter. Results (see Table 1 and Fig. 1) were presented
as percent inhibition of cell growth when compared to controls.
Plasmid DNA nicking and unwinding assays: Assays to evaluate
the effects of coumarins on plasmid DNA were performed as previously
described [30]. Briefly, 10 µL reaction mixtures were prepared that
contained 75 ng of plasmid pZeoSV DNA, 0.1 µL coumarin deriviative
solution (prepared as a 100 × concentrate in DMSO) and TE buffer
(10 mM Tris–HCl, 1 mM EDTA, pH 8). The reaction volume and com-
ponents were scaled up several-fold so that multiple aliquots of 10 µL
could be removed for analysis in agarose gels before and after UVA light
treatment. Treatment with UVA light was performed in V-bottomed 96-
well plates as described above for the cell cultures. Samples of the re-
action mixtures containing the plasmids were then analyzed by elec-
trophoresis using 1.2% agarose gels either in neutral gel buffer (40 mM
Tris base, 20 mM acetic acid, 1 mM EDTA) or under denaturing con-
ditions. To denature the DNA, samples were first mixed with 2 M NaOH
supplemented with 0.1 M EDTA and heated in a water bath at 90 °C for
1 min. After cooling on ice, DNA samples were loaded on 1.2% alkaline
agarose gels prepared and electrophoresed in alkaline gel buffer
(50 mM Tris base, 45 mM boric acid, 30 mM NaOH, 1 mM EDTA). After
electrophoresis, gels were washed and stained with ethidium bromide
(0.5 μg/mL). Gels were then photographed (see examples in Fig. 2) with
an Eagle Eye II digital documentation system (Stratagene, San Diego,
CA). Bands shown in Fig. 2 were then subjected to image analysis to
calculate the percent of plasmid unwinding (see Table 2) which ranged
from 0.2 to 99% in the set chosen.
182–183 °C, with elemental analysis (C,H,F,Cl within
0.3% of
theory) and 1H NMR in accord with theory. The same procedure for the
7-allyloxy compound (44) gave 34% of the rearranged 6-allyl-7-hy-
droxy (43) as off-white crystals, mp 174–175 °C. Elemental analysis
(C,H,F) was within
0.3% and 1H NMR was in accord with theory.
Acetylation of (43) with acetic anhydride/sodium acetate gave 58% of
the O-acetyl coumarin, compound (40), mp 151–152 °C (recrystalliza-
tion from MeOH). Analytical data confirmed structure and purity.
(18) 4,8-dimethyl-6-nitro-7-hydroxycoumarin. 4,8-Dimethyl-7-
hydroxycoumarin (12, 7.50 g, 39.4 mmol) was dissolved in 75 mL
concentrated sulfuric acid at room temperature, chilled to −20° C, and
treated to the addition of a pre-chilled nitrating mixture (3 mL con-
centrated HNO3 added to 9 mL concentrated H2SO4). Stirring was
continued for three hrs at −20 °C with the mixture allowed to warm
before pouring into ice. Bright yellow crystals were filtered, washed
with water and dried to recover 7.50 g (81% yield) crude solid. The
product (18) was recrystallized from ethanol to give yellow green
crystals with mp = 229.5–231.5 °C. C,H,N analysis was within
0.25% of theoretical. 1H NMR (DMSO‑d6): δ (ppm) 2.29 and 2.41 (two
s, 3H each, C4-CH3 and C8-CH3), 6.37 (s, 1H, C3H), 8.19 (s, 1H, C5H),
11.30 (br s, 1H, eOH). 13C NMR (DMSO‑d6): δ (ppm) 8.75, 18.00,
112.45, 112.93, 115.12, 119.90, 132.80, 152.91, 154.18, 155.10,
158.96.
(17) 4,8-Dimethyl-6-amino-7-allyloxycoumarin. 4,8-Dimethyl-
6-nitro-7-hydroxycoumarin (18, 4.70 g, 20 mmol) was added to
40 mL dry acetone with K2CO3 (2.76 g, 20 mmol) in an oven dried flask.
Allyl bromide (2.90 g, 24 mmol) in 40 mL of acetone was added slowly
with heating and stirring. After five hrs of vigorous agitation at reflux
the mixture was filtered and the solid on the filter washed with boiling
acetone. Evaporation of the filtrate gave 3.30 g (60%) of crude 4,8-
dimethyl-6-nitro-7-allyloxycoumarin which was employed directly
in the next step. To a round bottom flask was added finely ground 4,8-
dimethyl-6-nitro-7-allyloxycoumarin (3.00 g, 11 mmol), tin (3.24 g,
27.3 mmol), SnCl2 (3.04 g, 16.0 mmol) and 2 mL concentrated HCl in
200 mL ethanol. The mixture was stirred overnight during which all tin
dissolved. Most of the ethanol was removed in vacuo. As the solution
was allowed to cool, a gel formed which was filtered to recover a small
amount of pearlized crystals. Diethyl ether was added to the filtrate and
a precipitate formed which was filtered, and rinsed with ether to re-
move SnCl2. The solids were dried and extracted with hot ethanol on
the funnel of a filtration flask. Evaporation of the ethanol gave a crude
product that was recrystallized from ethanol to provide 2.20 g (82%) of
the 4,8-dimethyl-6-amino-7-allyloxycoumarin, (17). The bright
mustard yellow crystals had a mp of 145.5–147.5 °C. C,H,N analysis
within
0.30% of theoretical. 1H NMR (DMSO‑d6): δ (ppm) 2.28 and
5. Conclusions
2.49 (two s, each 3H, C4-CH3 and C8-CH3), 4.54 (d, 2H, -O-CH2-), 5.20
(br s, 2H, eNH2), 5.28 (d, 1H, =CH), 5.48 (d, 1H, =CH′), 6.16 (m, 1H,
eCH]), 6.39 (s, 1H, C3-H), 7.58 (s, 1H, C5-H). 13C NMR (DMSO‑d6): δ
Forty-nine 7-oxycoumarins from a library of 71 structural variants
demonstrated phototoxicity against PAM212 mouse keratinocytes.
8

C. Guillon, et al.
BioorganicChemistry89(2019)103014
None of the compounds was phototoxic in the absence of UVA light.
These data indicate that the ability of the compounds to inhibit cell
growth was dependent not only on their physiochemical characteristics,
but also on their ability to absorb UVA light. Based on the results, de-
finite structure-activity correlations could be made. Relative lipophili-
city was an important factor especially among structurally similar fa-
milies of coumarins. In most substituted oxycoumarins, the 7-alkoxys
were more active than the 7-hydroxy compounds. Coumarins with
electron withdrawing moieties – cyano and fluoro at C3 – were con-
siderably less active while those with bromines or iodine at that loca-
tion displayed enhanced activity. The electron withdrawing tri-
fluoromethyl at C4 was generally a deactivating functionality while
brominated alkyl side chains substantially improved activity.
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Of interest were the findings that photocytotoxic coumarins also
possessed the ability to photo-damage plasmid DNA albeit not in the
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There is no evidence that the two-ring coumarins, unlike the three-ring
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This work was funded in part by the National Institutes of Health
CounterACT Program through the National Institute of Arthritis and
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Its contents are solely the responsibility of the authors and do not ne-
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103014.
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