Sulfonyl-polyol N,N-dichloroamines with rapid, broad-spectrum antimicrobial activity

Article abstract of DOI:10.1016/j.bmcl.2013.08.027

The discovery and development of antimicrobial agents that do not give rise to resistance remains an ongoing challenge. Our efforts in this regard continue to reveal new potential therapeutic agents with differing physicochemical properties while retainin

Full text of DOI:10.1016/j.bmcl.2013.08.027

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5650–5653
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Sulfonyl-polyol N,N-dichloroamines with rapid, broad-spectrum
antimicrobial activity
⇑
Timothy P. Shiau , Eddy Low, Bum Kim, Eric D. Turtle, Charles Francavilla, Donogh J. R. O’Mahony,
Lisa Friedman, Louisa D’Lima, Andreas Jekle, Dmitri Debabov, Meghan Zuck, Nichole J. Alvarez,
Mark Anderson, Ramin (Ron) Najafi, Rakesh K. Jain
NovaBay Pharmaceuticals, 5980 Horton Street, Suite 550, Emeryville, CA 94608, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and development of antimicrobial agents that do not give rise to resistance remains an
ongoing challenge. Our efforts in this regard continue to reveal new potential therapeutic agents with dif-
fering physicochemical properties while retaining the effective N,N-dichloroamine pharmacophore as the
key antimicrobial warhead. In this Letter, we disclose agents containing polyol units as a water solubiliz-
ing group. These sulfonyl-polyol agents show broad spectrum bactericidal and virucidal activity. These
Received 12 July 2013
Accepted 5 August 2013
Available online 17 August 2013
Keywords:
Bactericidal
Virucidal
Bacteria
Virus
compounds show 1h MBC’s of 16–512
l
g/mL against Escherichia coli and 4–256
lg/mL against Staphylo-
coccus aureus at neutral pH, and 1-h IC50’s of 4.5–32
lM against Adenovirus 5 and 0.7–3.0
l
M against
Herpes simplex virus 1. The lead compounds were tested in a tissue culture irritancy assay and showed
only minimal irritation at the highest concentrations tested.
Ó 2013 Elsevier Ltd. All rights reserved.
Conjunctivitis
Chloroamines
Bacteria and viruses are quickly developing resistance to cur-
rently marketed drugs.1 To address this problem, an antimicrobial
agent with rapid bactericidal and virucidal activity and low poten-
tial for resistance development is desired. Based on N-chlorotau-
rine, a molecule produced by neutrophils,2 we have reported
bactericidal and virucidal activity of N,N-dichlorotaurine deriva-
tives with anionic3 as well as cationic4 solubilizing groups. We
are interested in further examining the role of the water-solubiliz-
ing group and structure–activity relationships of neutral solubiliz-
ers. In our continuing efforts, we report the discovery of agents
which are both potently bactericidal and virucidal and are excel-
lent candidates for the treatment of topical infections with un-
known etiology such as infectious conjunctivitis.
Conjunctivitis, commonly called ‘pink eye,’ is the inflammation
of the conjunctiva, and can arise from any one (or a combination)
of the following: infectious causes such as viruses (e.g., adenovirus,
herpesvirus, coxsackievirus, enterovirus)5 or Gram-positive or
Gram-negative bacteria (e.g., Staphylococcus aureus, Escherichia coli,
Streptococcus pneumoniae, Haemophilus influenzae),6 or noninfec-
tious causes such as chemical agents (e.g., sodium hydroxide, citric
acid) or allergic inflammation. Both viral and bacterial conjunctivi-
tis share many common symptoms, and there is great difficulty in
assessing what the causative agent actually is.7 Although most
cases of infectious conjunctivitis are viral,8 there are no approved
drugs for viral conjunctivitis. Practitioners who prescribe antibac-
terial agents as first-line treatments in these cases risk creating
drug-resistant bacteria for no clinical benefit.
An ideal drug to treat infectious conjunctivitis would have the
following characteristics: (a) be active against a broad spectrum
of bacteria, (b) be active against viruses, (c) applied topically to
the eye, (d) do not cause irritation, and (e) do not induce or give
rise to resistance even after multiple treatments.
Sulfone-extended backbone analogs previously reported with
anionic and cationic water-solubilizing groups prompted us to ex-
plore the possibility of neutral, polyol-solubilized analogs as a
means of increasing the clogP while maintaining water-solubility.
The key intermediate 3 was synthesized as previously reported3a
.
Alkylation of the sulfide with a haloalkanol or epoxide furnished
alcohols 4a–e, which upon oxidation with mCPBA, afforded sul-
fone-alcohols 5a–e (Scheme 1). The sulfone-alcohols were
N-deprotected by hydrogenation and chlorinated with tert-buty-
lhypochlorite to give compounds 1a–e. The enantiomers of 1e were
synthesized from enantiomerically pure glycidols and were tested
separately (see Tables 1 and 2).
Intermediate 3 was also alkylated with alkenes, which were in
turn dihydroxylated to the corresponding diols. Treatment of 3
with butadiene monoepoxide provided a 2:1 mixture of 6f (1° at-
tack of epoxide) and 6g (2° attack of epoxide), which were separa-
ble by silica gel chromatography. The alkylation of 3 by butene and
hexene derivatives to give 6h and 6i was straightforward. The
⇑
Corresponding author. Tel.: +1 (510)899 8862.
E-mail address: tshiau@novabaypharma.com (T.P. Shiau).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.027

T. P. Shiau et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5650–5653
5651
b
a
X
H
X
CbzHN
S
CbzHN
SH
CbzHN
S
n
O
5a-e
O
OH
3
4a-e
c
Cl
X
H
N
Cl
S
n
O
O
OH
1a-e
Scheme 1. Alkylation of intermediate 3 by alcohol-containing halides and epoxides. Reagents and conditions: (a) alkylating agent, Cs2CO3, DMF; (b) mCPBA, DCM, 0 °C; (c) H2
(1.3 atm), 10% Pd/C, MeOH; then t-BuOCl, MeOH, 0 °C.
Table 1
Two sulfonamide analogs 2a and 2b were synthesized through
the reaction steps as described in Scheme 6. Intermediate 13 was
Compounds synthesized in Scheme 1
oxidized with aqueous HOCl to afford sulfonyl chloride 14, which
was then reacted with N-methylaminoalcohols to give sulfona-
mides 15a–b. The sulfonamides were then N-deprotected and
N-chlorinated to give compounds 2a–b.
Prior to antimicrobial testing, all compounds were screened for
aqueous solution stability. In several cases, the compounds were
unstable in aqueous solution and were not tested for in vitro anti-
microbial activity. In one case (2b) the compound was stable in
aqueous solution but unstable as a solid.
Alkylating agent
X
n
Entries
I-(CH2)2OH
CH2
1
1
1
1
2
2
2
4a, 5a, 1a
4b, 5b, 1b
4c, 5c, 1c
4d, 5d, 1d
4e, 5e, 1e
R-4e, R-5e, R-1e
S-4e, S-5e, S-1e
Br-(CH2)3OH
Cl-CH2C(CH3)2CH2OH
Br-(CH2)8OH
( )-Glycidol
(R)-Glycidol
(S)-Glycidol
(CH2)2
CH2C(CH3)2
(CH2)7
CH2
CH2
CH2
Susceptibility testing of E. coli ATCC 25922 and S. aureus ATCC
29213 to 1a–m and 2a–b was conducted using modified CLSI
methods. CLSI M26-A protocol for minimum bactericidal concen-
tration (MBC) testing was modified by substituting 0.9% acetate
saline buffer, pH 4 (ASB) or phosphate-buffered saline, pH 7
(PBS) for Cation-Adjusted Mueller Hinton Broth (CAMHB) to cir-
cumvent the reactivity of chloroamine compounds to certain
components of CAMHB. To reflect the expected short exposure
time in ophthalmic formulations, the MBC assay was shortened
from 16–20 h at 35 °C to 1 h at room temperature. The MBC
was defined as the lowest concentration achieving >99.9% kill.
Microorganisms were first grown to mid-log phase, centrifuged
and suspended in ASB or PBS. Next, the organisms were added
to serial twofold dilutions of the test compound in ASB or PBS
to a final inoculum of 105–106 CFU/mL and incubated for 1 h at
room temperature.Aliquots of the cell suspension were plated
on agar with growth media, incubated 24 h at 35 °C and CFUs
were quantified.
Table 2
Compounds synthesized in Scheme 2
Alkylating agent
X
Entries
Butadiene monoepoxide
CH2CH(OH)
CH(CH2OH)
(CH2)2
6f, 7f, 1f
6g, 7g, 1g
6h, 7h, 1h
6i, 7i, 1i
Br-(CH2)2CH@CH2
Br-(CH2)4CH@CH2
(CH2)4
oxidation of both the alkene and the sulfide was accomplished
with a catalytic OsO4 procedure employing 3 equiv of NMO. Sul-
fone-polyols 7f–i were isolated, 7f and 7g as 4:1 and 2:1 mixtures
of diastereomers, respectively, which were not separated. Depro-
tection and N-chlorination afforded compounds 1f–i (Scheme 2).
Intermediate 3 can be alkylated with a wide range of polyhydroxy-
lated compounds. Alkylation with methyl 6-bromo-6-deoxy-a-D-
glucopyranoside afforded compound 8, which was oxidized to 9,
followed by deprotection and N-chlorination to give 1j (Scheme 3).
Two acetylated derivatives of 1e were synthesized by acetyla-
tion of intermediate 5e (Scheme 4). Treatment with 1 equiv acetic
anhydride afforded monoacetyl derivative 10k which was depro-
tected and N-chlorinated to give 1k, while an excess of acetic anhy-
dride afforded diacetyl derivative 10l which was deprotected and
N-chlorinated to give 1l.
Adenovirus 5 (Ad5, ATCC VR-1516) and Herpes simplex virus
1(HSV-1) (ATCC, strain F, VR-733) stocks were diluted 1:100 in
20 mM PBS or 5 mM ASB supplemented with 2.5% glycerol, ali-
quoted and stored at À80 °C. Threefold serial dilutions of com-
pounds were prepared in the respective buffer in 96 well plates
in quadruplicate. Freshly thawed virus was diluted 1:20 in the
same buffer, added 1:1 (v/v) to the diluted compounds and incu-
bated for 60 min at room temperature. Excess compound was neu-
tralized by adding the same volume of 2Â neutralization media
(2Â D-MEM/F12, Invitrogen, Carlsbad, CA) supplemented with
In previous studies,3b we have shown that a spacer consisting of
two methylenes between a sulfone and dichloroamine was ideal
for chemical stability. We also synthesized the one-methylene ana-
log of 1a to confirm this hypothesis. Previously reported interme-
diate 11 was alkylated, oxidized, deprotected, and N-chlorinated
in a sequence similar to that described in Scheme 1 for the synthe-
sis of 1a (from 3) to afford 1m (Scheme 5).
20% FBS, 1.2 g/L NaHCO3, 20 mM
L-glutamine and 1000 IU/mL pen-
icillin/100 g/mL streptomycin (Mediatech)) for 60 min at room
l
temperature. 200
l
L of the neutralized virus/compound mixtures
a
b
X
X
CbzHN
SH
Cl
CbzHN
S
CbzHN
S
OH
3
6f-i
O O
OH
7f-i
c
X
S
N
Cl
OH
O
O
OH
1f-i
Scheme 2. Alkylation of intermediate 3 with alkene-containing halides and epoxides. Reagents and conditions: (a) alkylating agent, Cs2CO3, DMF; (b) OsO4 (cat.), NMO,
acetone; (c) H2 (1.3 atm), 10% Pd/C, MeOH; then t-BuOCl, MeOH, 0 °C.

5652
T. P. Shiau et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5650–5653
b
O
OMe
OH
a
O
OMe
OH
CbzHN
S
CbzHN
S
CbzHN
SH
O
O
8
9
HO
3
HO
OH
OH
c
Cl
O
OMe
OH
N
Cl
S
O
O
HO
1j
OH
Scheme 3. Alkylation of intermediate 3 by a saccharide derivative. Reagents and conditions: (a) methyl 6-bromo-6-deoxy-
DCM, 0 °C; (c) H2 (1.3 atm), 10% Pd/C, MeOH; then t-BuOCl, MeOH, 0 °C.
a-
D-glucopyranoside, Cs2CO3, DMF; (b) mCPBA,
a
b,c
Cl2N
S
O
OR1
CbzHN
S
O
OR1
CbzHN
S
O
OH
O
O
OR2
OR2
O
OH
R1 = Ac, R2 = H (1k)
R1 = R2 = Ac (1l)
R1 = Ac, R2 = H (10k)
R1 = R2 = Ac (10l)
5e
Scheme 4. Acetylation of 5e and synthesis of acetylated analogs 1k and 1l. Reagents and conditions: (a) 1.2 equiv or 2.5 equiv Ac2O, pyridine, DMAP, DCM, 0 °C; (b) H2
(1.3 atm), 10% Pd/C, MeOH; then t-BuOCl, MeOH, 0 °C.
O
O
O
O
c
a,b
S
SH
Cl
S
CbzHN
11
CbzHN
OH
N
OH
Cl
12
1m
Scheme 5. Alkylation of intermediate 11 with iodoethanol. Reagents and conditions: (a) ICH2CH2OH, Cs2CO3, DMF; (b) mCPBA, DCM, 0 °C; (c) H2 (1.3 atm), 10% Pd/C, MeOH;
then t-BuOCl, MeOH, 0 °C.
Me
N
a
b
Cl
OH
n
CbzHN
SAc
CbzHN
CbzHN
S
S
14
Me
13
O O
O
O
n = 2 (15a)
n = 3 (15b)
c
Cl
N
OH
n
N
Cl
S
O
O
n = 2 (2a)
n = 3 (2b)
Scheme 6. Synthesis of sulfonamide derivatives 2a–b. Reagents and conditions: (a) HOCl, H2O, 0 °C; (b) MeHN(CH2)nOH; (c) H2 (1.3 atm), 10% Pd/C, MeOH; then t-BuOCl,
MeOH, 0 °C.
were added to A549 cells for Ad5 or Vero cells for HSV-1 (both
ATCC), which were prepared by seeding 5000 cells/well in a flat-
bottom 96-well plate on the day before the assay, and incubated
for 1–2 h at 37 °C to allow viral adsorption to the cells. Unbound
virus was aspirated. Cell culture medium without any compound
was added back to the cells. Cells were incubated for 6 days at
37 °C, 5% CO2, 90% relative humidity. Viral cytopathic effect (CPE)
was determined using Dojindo cell counting kit-8 (Rockville, MD)
and SpectraMax plate reader (Molecular Devices, Sunnyvale, CA).
Cytotoxicity controls without the addition of Ad5 were carried
out in parallel. The % inhibition of viral CPE was calculated as fol-
lows: (compound-treated infected sample—untreated infected
control)/(untreated, noninfected control-untreated, infected con-
trol) Â 100. The 50% inhibitory concentration (IC50) was calculated
by plotting (% inhibition) versus (logcompound concentration) in
GraphPad Prism 4 (La Jolla, CA) using the sigmoidal dose–response
equation with a top value constraint set to 100.
three-dimensional architecture and provides a nonanimal alterna-
tive to the traditional Draize rabbit eye test with excellent in vitro–
in vivo correlation.9 EpiOcular tissues (Mattek Corp.) were placed
in 900 lL cell culture media and 100 lL test compound was added
to the apical side of the tissue for varying exposure times. Tissues
were rinsed with 1Â PBS and placed in an MTT solution for 3 h. Tis-
sues were extracted overnight and tissue viability was determined
by MTT absorbance. Tissue viability was correlated with a Draize-
type score for tissue irritancy according to Mattek’s instructions.
Table 3 summarizes the antimicrobial activity as MBC values
against S. aureus and E. coli, and virucidal activity as IC50 values
against Ad5 and HSV-1. As expected, all tested compounds showed
excellent bactericidal activity at pH 4, most compounds with only
two to fourfold variations in activity. The only compound which
showed significantly different activity, 1j, has a molecular weight
much higher than the other compounds and the increased value
is simply a reflection of its higher molecular weight. All tested
compounds also showed excellent virucidal activity at pH 4, most
compounds with only twofold variations in activity.
The potential for ocular irritancy of Triton X-100 and compound
1e were compared. The EpiOcular™ tissue model has a cornea-like

T. P. Shiau et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5650–5653
5653
Table 3
Bactericidal and virucidal activities of reported compounds
Entry
pH 4
pH 7
g/ml)
MBC E. coli (
lg/ml)
MBC S. aureus (
lg/ml)
IC50 Ad5 (uM)
MBC E. coli (lg/ml)
MBC S. aureus (
l
IC50 Ad5 (uM)
IC50 HSV-1 (uM)
1a
1b
1c
1d
1e
R-1e
S-1e
1f
0.5
*
4
*
2
2
2
2
2
2
*
1
*
2
*
2
2
1
1
2
1
*
1.4
*
1.8
*
2.7
2.2
2.4
nt
1.5
1.4
*
8
*
16
*
128
128
4
4
4
32
16
16
*
4.5
*
0.7
*
64
256
16
32
32
128
64
64
*
9.6
32
26
14
13
nt
19
18
*
0.9
nt
1.5
1.2
1.0
nt
3.0
1.2
*
1g
1h
1i
1j
1k
1l
1m
2a
2b
8
2
1
1
2
*
8
4
2
1
1
*
nt
nt
nt
2.3
1.5
*
512
64
128
*
64
*
256
32
128
*
128
*
nt
nt
nt
*
14
*
nt
nt
nt
*
1.2
*
nt = not tested. ⁄ = not tested due to compound decomposition.
untreated control within 46 min, corresponding to a Draize score
of none or minimal irritancy (Fig. 1).
In summary, we have described the synthesis of sulfone-con-
taining analogs with polyols as neutral, water-solubilizing groups
for dichloroamines. These molecules have been found to have po-
tent virucidal activity against adenovirus 5 and herpes simplex
virus 1 in addition to bactericidal activity against S. aureus and E.
coli. These compounds show relatively little pH dependence com-
pared with anion- and cation-solubilized dichloroamines, having
potent activity at pH 7 in addition to pH 4, with compound 1e
showing the least pH dependence of the series. Compound 1e
was shown to be minimially irritating in an EpiOcular tissue mod-
el. Expanded preclinical work is being conducted to evaluate the
potential for these compounds to enter clinical trials for viral and
bacterial conjunctivitis.
Exposure time [min]
Acknowledgments
Figure 1. Effects of reported compound 1e on tissue viability in an EpiOcular
model.
The authors thank Professor John Soderquist and Dr. Larry
Truesdale for their help and support through these studies.
In contrast to the strong pH dependence seen for previously re-
ported compounds—256-fold increase in MBC for E. coli, 512-fold
increase in MBC for S. aureus, 22-fold increase in IC50 for Ad5—sev-
eral compounds showed minimal pH dependence between pH 4
and 7. Compound 1a showed a 16-fold increase in MBCs from pH
4 to pH 7 and a 3.2-fold increase in Ad5 IC50, while compound 1e
showed only a twofold increase in MBC for S. aureus and an eight-
References and notes
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.
.
These results lead us to hypothesize that the ability of com-
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pounds tested against HSV-1, a lipid-enveloped virus, showed 30-
fold improvement over other anionic compound (data not shown).
Compounds 1a and 1c show high nanomolar potency against HSV-
1 (Table 3).
7. American Academy of Ophthalmology Preferred Practice Pattern: Conjunctivitis.
Available
at:
http://one.aao.org/asset.axd?id=4848a02c-26e4-417f-8651-
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Mauger, T. Am. Fam. Physician. 2010, 15, 137.
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In the EpiOcular tissue irritancy model, 0.3% of compound 1e in
20 mM PBS pH 7 reduced the tissue viability to 50% of the