Synthesis and growth regulatory activity of a prototype member of a new family of aminothiol radioprotectors

Article abstract of DOI:10.1016/j.bmcl.2011.10.011

The synthesis, growth inhibition and radioprotective activity of the PrC-210 aminothiol, 3-(methylamino)-2-((methylamino)methyl)propane-1-thiol, and its polyamine and thiolated polyamine progenitors are reported. All of the molecules significantly inhibit

Full text of DOI:10.1016/j.bmcl.2011.10.011

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7426–7430
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis andgrowth regulatory activity of a prototype member of a new family
of aminothiol radioprotectors
Richard R. Copp b, Daniel D. Peebles b, William E. Fahl a,b,
⇑
a McArdle Laboratory for Cancer Research, University of Wisconsin Carbone Cancer Center, Madison, WI, USA
b ProCertus BioPharm, Inc., Madison, WI, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2011
Revised 29 September 2011
Accepted 4 October 2011
Available online 13 October 2011
The synthesis, growth inhibition and radioprotective activity of the PrC-210 aminothiol, 3-(methyl-
amino)-2-((methylamino)methyl)propane-1-thiol, and its polyamine and thiolated polyamine progeni-
tors are reported. All of the molecules significantly inhibited growth of cultured normal human
fibroblasts. The combination of an ROS-scavenging thiol group and a positively charged alkyl-amine
backbone provided the most radioprotective aminothiol molecule.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
3-(Methylamino)-2-
((methylamino)methyl)propane-1-thiol
Aminothiol
Growth regulator
Radioprotector
Polyamine
ROS-scavenger
Radiotherapy-induced dermatitis is a common side effect seen
in up to 85% of patients who receive a course of radiotherapy as
part of their cancer therapy regimen.1 A topically administered
radioprotector that could be applied prior to radiotherapy on each
of the 30 irradiation days would reduce pain and long term scar-
ring and would improve patient compliance in receiving all
30 days of treatment. Skipped radiotherapy days have a discernible
risk for the patient as measured by a decrease in 5 year survival
rate for breast cancer.2
There is also a need for new systemically administered radiopro-
tectors that lack the side effects of nausea/vomiting and hypoten-
sion/fainting that have hampered the use of current generation
aminothiol radioprotectors, most notably the five carbon amin-
othiophosphonate, amifostine.3
The aminothiol radioprotector design concepts of: (i) a flexible
alkyl chain backbone, which carries a positive charge due to one
or more amine groups, to achieve ionic interaction and concentra-
tion around negatively charged DNA in cells, and (ii) the presence
of a free or capped thiol group to scavenge oxygen free radicals
formed from ionizing radiation, have been used before in programs
to build radioprotective molecules within both the U.S.4 and the
former Soviet Union.5
In the present investigation, we disclose a process in which: (i)
the number of alkyl-amine segments in the aminothiol backbone is
systematically increased to increase drug–DNA affinity and ionic
interaction, resulting in increased growth inhibition that is associ-
ated with this enhanced drug–DNA interaction, and (ii) the place-
ment or ‘display’ of a free thiol reactive oxygen species (ROS)
scavenger at the end of a short alkyl side chain that displaces or
‘displays’ the scavenger moiety away from the DNA backbone to
theoretically enable ROS scavenging before ROS attack on dG bases
within cellular DNA. This work has resulted in a small family of
new aminothiol molecules,6,7 the prototype of which, PrC-210, is
described in initial detail here.
The synthesis of PrC-210, shown in Scheme 1, started with a
double displacement of chloride from 1 using N-methyl mesitylene-
sulfonamide (2) and sodium hydride, to form allylic sulfonamide 3.
Hydroboration of 3 afforded clean conversion to sulfonamide
alcohol 4. Using standard conditions, 4 was converted to mesylate
5 which was immediately treated with potassium thioacetate to
form 6. Following an established procedure,8 the mesitylene (Mts)
protecting groups were removed with HBr/HOAc, in the presence
of excess phenol. The deblocking procedure also hydrolyzed the
thioacetate group. Work up resulted in a mixture of PrC-210 and
the corresponding disulfide (dimer). The mixture was treated with
2-mercaptoethanol to cleave the disulfide and the product, PrC-
210, was precipitated from EtOH as the HCl salt. Subsequent recrys-
tallizations removed the sulfurous odor.
⇑
Corresponding author. Address: 1400 University Avenue, Madison, WI, USA.
Tel.: +1 608 262 1275; fax: +1 608 262 2824.
E-mail addresses: fahl@oncology.wisc.edu, fahl@procertus.com (W.E. Fahl).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.011

R. R. Copp et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7426–7430
7427
OH
N
a
b
c
Mts
N
N
+
N
Mts
Mts
H
Cl
Cl
N
N
3
2
4
Mts
1
Mts
Mts = mesitylenesulfonyl
OMs
d
SAc
SH
e, f, g
N
HN
Mts
Mts
2HCl
N
N
NH
Mts
Mts
5
6
PrC-210
Scheme 1. Reagents: (a) NaH, DMF, THF; (b) BH3–THF; EtOH, H2O2, NaOH (aq); (c) MsCl, Et3N, CH2Cl2; (d) KSAc, DMF; (e) HBr, PhOH, HOAc, CH2Cl2; (f) K2CO3 (aq); (g) HCl
(aq), 2-mercaptoethanol, EtOH; recrystallize.
O
a
b
NPht
NPht
NK
NH
+
2
Cl
Cl
NH
2
2HCl
O
7
1
8
9
OH
OMs
c
d
e
f
NHMts
NHMts
NHMts
11
NHMts
NHMts
NHMts
12
10
SH
SAc
g, h, i
NH
2
NHMts
NH
2HCl
2
NHMts
PrC-211
13
Scheme 2. Reagents: (a) 2 equiv potassium phthalimide, DMF; (b) (1) NH2NH2–H2O, (2) HCl; (c) NaOH, mesitylenesulfonyl chloride; (d) (1) BH3–THF, (2) H2O2, NaOH; (e)
MsCl, Et3N, CH2Cl2; (f) KSAc, DMF; (g) HBr, PhOH, HOAc, CH2Cl2; (h) K2CO3 (aq); (i) HCl (aq), 2-mercaptoethanol; recrystallize.
The synthesis of PrC-211, shown in Scheme 2, employed a mod-
ification of the route used for PrC-210. An attempt was made to form
sulfonamide 10 directly by displacement of chloride from 1 using
mesitylenesulfonamide, activated with sodium hydride. A complex
mixture formed, from which 10 could not be isolated in pure form.
Alternatively, 1 was treated with potassium phthalimide to form
allylic phthalimide 8.9 Removal of the phthalate groups with hydra-
zine provided 9, which upon treatment with mesitylenesulfonyl
chloride afforded the bis-sulfonamide 10 in good yield. Using the
sequence from the PrC-210 preparation, hydroboration, mesylation,
thioacetate displacement and deblocking, PrC-211 was obtained as
the HCl salt and subsequently recrystallized.
The amine side chains, synthesized according to the route
illustrated in Scheme 3, were constructed as sulfonamide-protected
intermediates, each with a single point of attachment (N–H), at one
terminus, for coupling to the olefinic core (Fig. 1E). Preparations for
sulfonamides 17 and 20 have previously been described.10 A conve-
nient alternative approach was found that employed a modification
of a reported method,11 starting with N-(4-bromobutyl)phthali-
mide (14). Displacement with N-ethyl mesitylenesulfonamide and
sodium hydride, afforded 15, which upon treatment with hydrazine,
was converted to 16. Amidation with mesitylenesulfonyl chloride,
under standard conditions, gave 17. Sulfonamide 17, a protected
diamine, represents the smallest amine side chain unit in the series,
which includes 20, 21, and 22. Sulfonamide 17 was systematically
elaborated to the subsequent side chain units using the same meth-
odology as described for 17.
Coupling of the various amine side chains to the olefinic core is
shown in Scheme 4. The synthesis commenced with the known
TBS-protected allylic alcohol 23.12 Mesylation, using standard con-
ditions, provided the activated intermediate 24. Coupling with a
sulfonamide side chain and sodium hydride provided 25. Removal
of the TBS-protecting groups with HCl afforded diol 26. Treatment
of 26 with 1 equiv of benzoyl chloride, in the presence of pyridine,
afforded alcohol 27 as a mixture of isomers. Activation of the allylic
alcohol 27, for amine side chain coupling, was attempted with
methanesulfonyl chloride, resulting in a complex mixture. Alterna-
tively, the alcohol was converted to bromide 28, with phosphorous
tribromide, and immediately coupled with a second side chain unit
to form 29. Hydrolysis of the benzoate group was carried out under

7428
R. R. Copp et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7426–7430
b
a
c
NH
NPht
2
NPht
N
N
Br
Mts
Mts
16
14
15
Mts
N
H
d
b
N
N
Mts
N
NPht
Mts
Mts
18
17
Mts
N
Mts
N
c
Mts
N
Mts
N
H
N
Mts
NH
2
20
19
Mts
N
d, b, c
H
N
d, b, c
N
N
Mts
Mts
Mts
21
Mts
N
Mts
N
Mts
N
Mts
N
Mts
N
H
22
Scheme 3. Reagents and conditions: (a) N-ethyl mesitylenesulfonamide, NaH, DMF; (b) N2H4–H2O; (c) mesitylenesulfonyl chloride, Et3N, CH2Cl2; (d) 4-bromobutylph-
thalimide, NaH, DMF.
C
A
NH
NH
HN
HN
HN
HN
HN
HN
NH
NH
HN
HN
NH
NH
NH
NH
HN
HN
NH
NH
HN
HN
HN
HN
HN
NH
HN
HN
NH
NH
HN
NH
µ
B
SH
D
E
HN
NH
NH
NH
SH
SH
HN
HN
HN
HN
NH
NH
HN
HN
HN
NH
NH
NH
NH
HN
HN
HN
NH
NH
NH
SH
SH
HN
HN
NH
HN
HN
NH
NH
*
*
*
Figure 1. Growth inhibition of normal human cells in culture by polyamine and aminothiol molecules. Structures of (A) first generation synthetic polyamines. (B) First
generation ‘thiolated polyamines’ and the next generation aminothiol, PrC-210. (C) Molecules were added at indicated concentrations to log phase, growing cultures of
diploid human fibroblasts, and cell numbers were measured 4 days later. (D) Growth inhibition is directly related to the number of [NH–(CH2)] segments present in the
polyamine or aminothiol molecule. (E) Olefinic core—common to polyamine and polyamine-thiol series.

R. R. Copp et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7426–7430
7429
R
Mts
N
b
a
c
OMs
OH
OTBS
TBSO
OTBS
TBSO
TBSO
OTBS
24
23
25
R
Mts
R
Mts
R
Mts
Mts
N
R
N
N
N
e
f
d
OBz
R
N
HO
OBz
Br
OBz
HO
OH
Mts
29
26
27
28
Mts
g
N
OH
Allyl Polyamine
Alcohol Intermediate
R
R
N
Mts
30
Scheme 4. Reagents: (a) MsCl, Et3N, CH2Cl2; (b) amine side chain, NaH, DMF; (c) HCl, MeOH; (d) BzCl, Pyd; (e) PBr3, toluene; H2O; (f) amine side chain, NaH, DMF; (g) NaOH.
SAc
Mts
R
Mts
R
Mts
R
d
a
N
OMs
N
N
OH
R
R
R
N
N
N
34
Mts
Mts
31
Mts
30
b
c
Mts
SH
N
Mts
R
HN
R
N
R
NH
R
N
35
32
Mts
Allyl Polyamine Thiols
c
HN
HN
R
Allyl Polyamines
R
N
H
33
Scheme 5. Reagents: (a) MsCl, Et3N, CH2Cl2; (b) N-Et mesitylenesulfonamide, NaH, DMF; (c) HBr, PhOH, HOAc, CH2Cl2; K2CO3 (aq); HCl (aq); (d) KSAc, DMF.
standard conditions to afford the versatile polyamine alcohol inter-
mediate 30, which was a common intermediate for the formation
of both polyamines and polyamine thiols.
Schemes 3–5. We anticipated that these drug molecules would
provide radio- and chemo-protection to human cells by inducing
a cell cycle block in mammalian cells at the G1/S cell cycle border
because of tight binding of the (+) charged polyamine backbones to
the (À) charged DNA backbone.13 Such a block can provide time for
DNA repair in radiation- or mutagen-treated cells before washout
of the polyamine drug and restoration of cell cycle progression.14
Figure 1B shows a nearly identical set of polyamines now with
the addition of a thiol group to each molecule. The goal in synthe-
sizing these molecules was to achieve the same cell cycle inhibi-
tion anticipated for the Figure 1A molecules, but now combined
with the addition of a thiol group that could serve as a scavenger
for the burst of short-lived ROS that is generated when mammalian
tissue is irradiated.15 The aminothiol and polyamine structures in
Figure 1B are growth inhibitory when added to rapidly growing,
normal human fibroblasts, and the potency of growth inhibition
Conversion of the versatile intermediate 30 to polyamines and
polyamine thiols is shown in Scheme 5. Treatment of 30 with meth-
anesulfonyl chloride and triethylamine afforded the mesylate 31.
Displacement with N-ethyl mesitylenesulfonamide, activated with
sodium hydride, provided 32 which was deblocked with HBr/HOAc
and phenol, to afford crude polyamine 33. Free-basing with potas-
sium carbonate followed by HCl treatment gave 33 hydrochloride
salt as a mixture of cis/trans isomers. Mesylate intermediate 31
was treated with potassium thioacetate to form 34 which was de-
blocked, using the same method as for 32, to form 35. Free-basing
and treatment with HCl provided 35 as the hydrochloride salt.
Figure 1A provides a summary of the first set of polyamine
structures that was synthesized using the strategies outlined in

7430
R. R. Copp et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7426–7430
times in the 2 h before irradiation, and control rats received only
SH
topical vehicle before irradiation (Fig. 2). Additional drug/irradia-
tion groups of three rats each received either the alkyl-thiol moiety
or the alkyl-diamine moiety from PrC-210 in the same delivery
vehicle. Following the 17.33 Gy irradiation of a 1.5 Â 3.0 cm patch
of dorsal skin on the rats’ backs, rats were scored 13 days later. The
ability of the PrC-210 aminothiol to completely prevent radiation
dermatitis was striking. Other radiodermatitis test groups explor-
ing efficacy of thiolated polyamines or other PrC-200 series amino-
thiols have shown that PrC-210 is the most potent and most
effective topical aminothiol to date.
Topical
Vehicle
N
H
N
H
SH
N
H
N
H
No
Radiation
PrC-210
Acknowledgements
This work was supported by Grant #CA-22484 from NCI as well
as research funds from ProCertus BioPharm, Inc.
Topical Treatments Prior to Irradiation
Figure 2. Prevention of radiation-induced dermatitis in rat model by prior topical
application of the PrC-210 aminothiol or its component structures. Molecules were
applied to skin in an alcohol/water delivery vehicle prior to receiving a 17.33 Gy
radiation dose to the 1.5 cm  3.0 cm skin site. Radiation dermatitis severity (i.e.,%
of the site covered by scab material) was scored 13 days following irradiation.
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was directly related to the number of (NH–(CH2)n) segments pres-
ent within the molecule (Fig. 1D).
Early in vitro studies of radioprotection with cultured cells dem-
onstrated that the long, polyamine structures were so growth-inhib-
itory (Fig. 1C) that we could not add sufficient moles of the thiolated
polyamines (e.g., PrC-117) to cell cultures that would enable the
thiol groups to significantly scavenge and radioprotect when the
tissue culture cells were irradiated. This provided the impetus to de-
sign and synthesize the PrC-200 series of small aminothiols, of
which PrC-210 is the prototype.
To determine if PrC-210 could function as a topical radioprotec-
tor that could prevent radiation dermatitis when applied prior to a
cancer patient’s daily radiotherapy, a rat assay that realistically
mimics human radiation dermatitis was created.16 This assay
quantifies the severity of radiation dermatitis in rat skin 13 days
after a single radiation dose of 17.33 Gy. In this study, PrC-210
was applied to rat skin in an ethanol/water delivery vehicle four
15. Leach, J. K.; Van Tuyle, G.; Lin, P.-S.; Schmidt-Ullrich, R.; Mikkelson, R. B. Cancer
Res. 2001, 61, 3894.
16. Peebles, D. D.; Soref, C. M.; Copp, R. R.; Fahl, W. E. Radiat. Res. (in press).