N,N-Dichloroaminosulfonic acids as novel topical antimicrobial agents

Article abstract of DOI:10.1016/j.bmcl.2008.10.114

2-Dichloroamino-2-methyl-propane-1-sulfonic acid sodium salt (2a), a stable derivative of endogenous N,N-dichlorotaurine (1), has been identified and is under development as a topical antimicrobial agent. Structure-activity relationships of analogs were explored to achieve optimal antimicrobial activity with minimal mammalian toxicity while maintaining the desired stability. All the analogs synthesized showed antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida albicans in the range of 1-128 μg/mL and cytotoxicity against mammalian L929 cells in the range 80-1900 μg/mL.

Full text of DOI:10.1016/j.bmcl.2008.10.114

Bioorganic & Medicinal Chemistry Letters 19 (2009) 196–198
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N,N-Dichloroaminosulfonic acids as novel topical antimicrobial agents
Eddy Low, Satheesh Nair, Timothy Shiau, Barbara Belisle, Dmitri Debabov, Chris Celeri, Meghan Zuck,
*
Ron Najafi, Nafsika Georgopapadakou, Rakesh Jain
NovaBay Pharmaceuticals Inc., 5980 Horton Street, Suite 550, Emeryville, CA 94608, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Dichloroamino-2-methyl-propane-1-sulfonic acid sodium salt (2a), a stable derivative of endogenous
N,N-dichlorotaurine (1), has been identified and is under development as a topical antimicrobial agent.
Structure–activity relationships of analogs were explored to achieve optimal antimicrobial activity with
minimal mammalian toxicity while maintaining the desired stability. All the analogs synthesized showed
antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida albicans in the range of
Received 7 October 2008
Revised 20 October 2008
Accepted 27 October 2008
Available online 31 October 2008
1–128 lg/mL and cytotoxicity against mammalian L929 cells in the range 80–1900 lg/mL.
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
N-Chloroamines
Antimicrobial agents
Taurine
Bacteria are increasingly resistant to most currently available
antibiotics. To meet the continuing need for antimicrobial agents
with novel mechanisms of action and low potential for develop-
ment of resistance we initiated a program for the development of
N-chlorotaurine-based molecules as topical antimicrobial agents.
Taurine (2-aminoethanesulfonic acid) is a conditionally essen-
tial amino acid known to have various physiological functions.1
N-chlorotaurine (3) and N,N-dichlorotaurine (1) are produced from
taurine during the respiratory burst in activated neutrophils and
macrophages2 via the scavenging of myeloperoxidase-produced
hypochlorous acid.
was identified5 as a stable analog of 1, exhibiting a half life of
>2 years at 40 °C and provided impetus to further develop this
class of compounds.
We expected that our initial lead, 2a, could be further optimized
for topical antimicrobial potency, in vivo efficacy and cytotoxicity
by suitable structural modifications (Fig. 1). Here, we report the de-
sign, synthesis, and biological activity of various backbone modifi-
cation and sulfonic acid replacements in 2a.
H H
N
H
H H
N
Cl
Me Me
N
Cl
β
-modifications
Cl
SO3Na
Cl
SO3Na
Cl
SO3Na
Me Me
SO3H
R R
acid
isosteres
SO3H
Cl2N
Cl2N
3
2a
1
backbone modifications
Nagl et al.3 previously described the bactericidal, fungicidal and
virucidal activity of N-chlorotaurine. Due to their non-specific
mechanism of action, this class of compounds has low potential
for the development of resistance. Despite potent antimicrobial
activity and low cytotoxicity, therapeutic use of N-chlorotaurine
is limited by its poor long-term solution stability at room temper-
ature.4 We believed N,N-dichlorotaurine (1) would be more stable
but discovered it still lacked the long-term stability required for
therapeutic agents. We presumed the transient nature of 1 was
due to rapid dehydrochlorination and introduced a dimethyl group
at the b-carbon to block this transformation. Thus compound 2a
Figure 1. SAR strategy on N,N-dichlorotaurine 2a.
a,b
O2N
CO2Me
SO3H
Boc-HN
Boc-HN
CO2Me
85%
4
5
c 71%
d-g
Cl
N
OH
15%
Cl
2b
6
Scheme 1. Reagents and conditions: (a) AcOH, 10% Pd–C, H2, 16 h; (b) Boc2O,
CH2Cl2, Et3N, 24 h; (c) LiBH4, THF, 0–25 °C, 16 h; (d) MeSO2Cl, Et3N, CH2Cl2, 0 °C, 2 h;
(e) 4 M HCl/dioxane, 16 h; (f) aq 1 M Na2SO3, 25 °C, 16 h; (g) aq HOCl, 5–10 °C, 1 h.
* Corresponding author. Tel.: +1 510 899 8871; fax: +1 510 740 3469.
E-mail address: rjain@novabaypharma.com (R. Jain).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.114

E. Low et al. / Bioorg. Med. Chem. Lett. 19 (2009) 196–198
197
a-c
d-g
Cl
Cl
CO2H
N3
Z-HN
OMs
N
j
SO3H
O
53%
34%
7
8
2c
h,b
73%
O
i
e,f,
Cl
CO2H
SAc
SO3H
Z-HN
Z-HN
O
10
N
O
2d
41%
24%
Cl
9
Scheme 2. Reagents and conditions: (a) LiAlH4, ether, 0–25 °C, 16 h; (b) Z-OSu, isopropanol–H2O, 16 h; (c) MeSO2Cl, Et3N, CH2Cl2, 0 °C, 2 h; (d) KSAc, DMF, 25 °C, 16 h; (e)
HCO2H, 30% H2O2, 25 °C, 16 h; (f) MeOH, 10% Pd–C, H2, 12 h, 25 °C; (g) aq HOCl, 5–10 °C, 1 h; (h) 10% Pd–C, H2, HCO2H, 12 h, 25 °C; (i) 2-(Acetylthio)ethanol, CDI, CH3CN, 70 °C,
16 h; (j) tert-Butyl hypochlorite, MeOH, 0–25 °C, 2 h.
Cl
Cl
O
SO3H
N
O
2e
46%
P = Z
P
OH
Z
O
Cl
e,b,c,f Cl
O
SO3H
N
H
N
H
N
d, 63%
51%
O
Cl
O
2f
13
11
h
-
l
Cl
O
SO3H
N
Cl
Cl
SO2NRMe
61%
g
63%
N
P = Z
2g
Cl
2i (R = H), 65%
2j (R = Me), 56%
c,f
SO2Cl
Z N
H
Z
SAc
14
Cl
N
SO2NHAc
N
m
51%
H
-
o
O O
S
12
Cl
,
b
,
c
Cl
2k
,
f
N
Cl
SO3H
53%
2h
Scheme 3. Reagents and conditions: (a) 3-(Acetylthio)propanoic acid, CDI, DMF, 25 °C, 16 h; (b) HCO2H, 30% H2O2, 25 °C, 16 h; (c) MeOH, 10% Pd–C, H2, 25 °C, 24 h; (d)
3-Chloro-2,2-dimethylpropanoyl chloride, Et3N, CH2Cl2, 25 °C, 16 h; (e) DMF, KSAc, 80 °C, 3 h; (f) tert-Butyl hypochlorite, MeOH, 0–25 °C, 2 h; (g) AcSH, DIAD, PPh3, THF, À5 to
25 °C; (h) DMF, NaH, allyl bromide, 0–25 °C, 16 h; (i) 9-BBN, THF, 25 °C,16 h; (j) MeSO2Cl, Et3N, CH2Cl2, 0 °C, 2 h; (k) 4 M HCl/dioxane, 25 °C, 16 h, aq 1 M Na2SO3, 40 °C, 16 h;
(l) aq HOCl, 5–10 °C, 1 h; (m) MeOH–MeONa, 25 °C, 4 h; (n) 1-bromo-2-chloroethane, Cs2CO3, DMF, 25 °C, 16 h; (o) KSAc, DMF, 70 °C, 16 h; (p) HOCl, H2O, CH2Cl2, 0.5 h; (q)
40% aq MeNH2, 5–25 °C, 3 h; (r) 40% aq Me2NH, 0–25 °C, 3 h; (s) 30% aq NH3, 5–25 °C, 3 h; (t) CH2Cl2, Ac2O, DIPEA, 25 °C, 16 h.
Our first course of action was to synthesize analogs with
b-modifications. This series of analogs were surprisingly unstable
and was presented elsewhere.6
The synthesis of N,N-dichloroamine 2b began with the key
intermediate 5, prepared from methyl 4-methyl-4-nitropentanoate
4 as shown in Scheme 1. The alcohol in 6 was converted into the
sulfonic acid group in two steps, through the mesylate intermedi-
ate which was displaced with Na2SO3 to yield the sulfonic acid. The
final chlorination was achieved with hypochlorous acid (formed
in situ from sodium hypochlorite under acidic pH) to furnish the
N,N-dichloro compound 2b.
The chloramine 2c was synthesized from azide 77 following the
sequence of steps illustrated in Scheme 2. LAH reduced both the
acid and the azido functionality in 7 to the amino alcohol, which
was Z-protected and converted to the mesylate 8. Reaction with
sodium sulfite and chlorination as described above afforded 2c.
We also introduced various functional groups in the backbone
to gain an understanding of the tolerance of these groups to the
O
S
O
S
a
H
N
j,a,g,i
57%
PO3Et2
Cl
Cl
NH2
SO3H
SO3H
N
N
H2N
N
N
29%
H
O
15
2l
17
18
b (R = H) or
c,d (R = Et)
34%
a-e
O
O
P
Me
N
H
N
OR
OR
f-i
e
Cl
P
Cl
Cl
N
H2N
Z-HN
Me
OH
OH
45%
Cl
O
2n (R = Et)
19 (R = Et)
20 (R = H)
16
2m
30 %, 3 steps
2o
(R = H)
46 %, 2 steps
Scheme 4. Reagents and conditions: (a) Boc2O, THF, À40 to 25 °C, 16 h; (b) Z-OSu,
isopropanol–H2O, 16 h, (c) 4 M HCl/dioxane, 25 °C, 16 h; (d) HCO2H, CDI, DMF,
25 °C; (e) BH3ÁMe2S, THF, 0–25 °C, 16 h, 1 M MeOH–HCl; (f) 3-(Acetylthio)propanoic
acid, CDI, DMF, 70 °C, 16 h; (g) HCO2H, 30% H2O2, 16 h; (h) MeOH, 10% Pd–C, H2,
16 h; (i) MeOH, tert-Butyl hypochlorite, 25 °C, 1 h; (j) 3-(Acetylthio)propanoic acid,
CDI, THF, À70 °C to rt, 4 h.
Scheme 5. Reagents and conditions: (a) n-BuLi, TMEDA, THF, MePO3Et2, À78 °C,
4 h; (b) TMSBr, CH3CN, 65 °C, 1 h; (c) NaOH, EtOH–H2O, 80 °C, 12 h; (d) MeOH, 4 M
HCl/dioxane, 25 °C, 1 h; (e) aq HOCl, 5–10 °C, 1 h.

198
E. Low et al. / Bioorg. Med. Chem. Lett. 19 (2009) 196–198
Table 1
tected diamine 16 in four steps as shown in Scheme 4. The sulfonic
Biological activity of compound 2a and its analogs.
acid group of compound 2a was also replaced with acid isosteres
such as phosphonates. Phosphonate analogs 2n and 2o were pre-
pared from the sulfinimine 1711 as illustrated in Scheme 5. Addi-
tion of ((diethoxyphosphoryl)methyl) lithium to 17 provided 18,
which on selective hydrolytic conditions (Scheme 5) gave 19 or
20. Chlorination using HOCl provided the desired dichloramines
2n and 2o.
The data in Table 1 summarizes the antimicrobial activity for all
analogs with sufficient aqueous solution stability (>24 h at room
temperature). The analogs are active against all organisms tested,
with no significant difference between the in vitro activities for
Gram-positive versus Gram-negative organisms. Activity against
Candida albicans was the most variable for the compounds tested,
ranging from 4 ug/mL in the case of compound 2e to greater than
Compound
MBC or MFCa
(l
g/mL)
CT50
(
l
g/mL)
t pH 4
½
Mouse fibroblast (saline)
L929 cells
pH 4 (saline)
S. aureus
E. coli
C. albicans
(days)
ATCC 29213 ATCC 25922 ATCC 10231
2a
2b
2c
2d
2e
2f
2h
2k
2n
2o
1b
8b
4
4c
8c
4
2
2
4
4
2
ND
8
32d
>128d
16
1200
640
1900
270
130
840
1820
ND
>200
16
174
>95
64
80
>95
44
2
64
1b
8
4d
16
64
8
1
64
2b
16
32d
16
80
130
>18
23
a
MBC is determined using a modification of a standard method described in CLSI
M26-A where Mueller–Hinton broth (MHB) is replaced by isotonic saline at pH 4 to
compensate for the reactivity of chlorine to certain components of MHB. Due to the
rapid cidal nature of chlorinated derivatives the assay was shortened from 16 to
20 h at 35 °C to 1 h at room temperature.
128 lg/mL in the case of compound 2b. In terms of cytotoxicity,
the phosphonate analogs, 2n and 2o, as well as the reverse ester
2e had the highest in vitro toxicity, about 10-fold higher than the
lead compound 2a; however all compounds had therapeutic indi-
ces (ratio of CT50 to MBC) from 8 to 1200 for bacteria and from 2
to 118 for C. albicans. Since the antimicrobial activity of these mol-
ecules is due to the oxidative capacity of the dichloramine func-
tionality, we did not observe any significant SAR among the
analogs in this class.
In summary, we have described the synthesis and antimicrobial
activity of various analogs of N,N-dichloramino-2-methylpropane-
1-sulfonic acid 2a. Diverse functional groups have been identified
that provide stability to the molecules as well as groups that are
tolerant to the dichloramine functionality. These molecules have
been evaluated as backups for our lead clinical candidate 2a.
b
S. aureus MCC 91731.
E. coli MCC 80392.
C. albicans MCC 50319.
c
d
chloramino functionality. The ester analog 2d (Scheme 2) was
prepared from the acid 9, which was obtained by the hydrogena-
tion of azide 7 using Pd–C in formic acid. Coupling of 9 with S-2-
hydroxyethyl ethanethiolate under CDI8 coupling conditions gave
the intermediate thioacetate 10. Oxidation of 10, followed by
N-deprotection and chlorination using tert-butyl hypochlorite
afforded the final dichloramine 2d.
We next introduced a range of functional groups in the back-
bone of 2a as depicted in Scheme 3. The protected amino alcohol
11 and its thioacetate derivative 12 served as common starting
materials for most of these analogs. Thus, the coupling of the ami-
no alcohol 11 with 3-(acetylthio)propionic acid9 under CDI condi-
tions afforded an intermediate thioacetate, which was oxidized to
sulfonic acid using HCO2H–H2O2. N-Chlorination using HOCl as
before yielded the desired reverse ester analog 2e. The sterically
hindered ester 2f was also synthesized from 11 and 3-chloro-2,2-
dimethylpropanoyl chloride. The coupled ester 13 on reaction with
potassium thioacetate followed by oxidation gave the sulfonic acid.
N-Deprotection and chlorination using tert-butyl hypochlorite
yielded the N,N-dichloro analog 2f (Scheme 3).
The ether-linked and sulfone-linked analogs 2g and 2h, respec-
tively, were also synthesized (Scheme 3). The ether analog 2g was
synthesized from 11 following reaction steps as shown in Scheme
3. Allylation of 11 with allyl bromide followed by hydroboration,
mesylation, displacement with sodium sulfite, and chlorination
with HOCl afforded 2g. The sulfone 2h was accessed from thioace-
tate 12, obtained by a direct Mitsunobu reaction10 between the
alcohol 11 and thioacetic acid followed by reaction sequences
shown in Scheme 3.
Several sulfonic acid replacements, like sulfonamide and N-
acetyl sulfonamide analogs, were also synthesized from thioace-
tate intermediate 12. Treatment of 12 with hypochlorous acid led
to the formation of the corresponding sulfonyl chloride 14, which
was reacted with methylamine or dimethylamine to afford 2i
and 2j, respectively after N-deprotection and chlorination using
tert-butyl hypochlorite. Similarly, the use of ammonia provided
the primary sulfonamide, which was treated with acetic anhydride
followed by N-deprotection and chlorination as described for the
preparation of 2i and 2j, gave 2k.
Acknowledgments
The authors acknowledge Dr. Behzad Khosrovi, Dr. Charles
Francavilla, and Bum Kim for useful discussions, Dr. Michael
Flagella and Nichole Alvarez for technical assistance on biological
assays and Dr. Lu Wang, Ashley Houchin, and Ping Xu for analytical
support.
References and notes
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Amide-linked analogs 2l and 2m were prepared from commer-
cially available building block 15 (Scheme 4). Coupling of 15 with
3-(acetylthio)propanoic acid under CDI conditions followed by oxi-
dation and N-chlorination as described earlier yielded compound
2l. Similarly, compound 2m was synthesized from the monopro-
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