An improved synthesis of haloacetamidine-based inactivators of protein arginine deiminase 4 (PAD4)

Article abstract of DOI:10.1016/j.tetlet.2008.05.021

Protein arginine deiminase 4 (PAD4) is an enzyme that hydrolyzes peptidyl arginine residues to form citrulline and ammonia. This enzyme has been implicated in several disease states, for example, rheumatoid arthritis, and therefore represents a unique target for the development of a novel therapeutic. A solution-phase synthesis of Cl-amidine, the most potent PAD4 inactivator described to date, has been developed. This synthesis proceeds in 80% yield over four steps at a significantly (12-fold) lower cost.

Full text of DOI:10.1016/j.tetlet.2008.05.021

Tetrahedron Letters 49 (2008) 4383–4385
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Tetrahedron Letters
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An improved synthesis of haloacetamidine-based inactivators of protein
arginine deiminase 4 (PAD4)
*
Corey P. Causey, Paul R. Thompson
Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, SC 29208, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein arginine deiminase 4 (PAD4) is an enzyme that hydrolyzes peptidyl arginine residues to form
citrulline and ammonia. This enzyme has been implicated in several disease states, for example, rheuma-
toid arthritis, and therefore represents a unique target for the development of a novel therapeutic. A solu-
tion-phase synthesis of Cl-amidine, the most potent PAD4 inactivator described to date, has been
developed. This synthesis proceeds in 80% yield over four steps at a significantly (12-fold) lower cost.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 5 March 2008
Revised 1 May 2008
Accepted 5 May 2008
Available online 9 May 2008
Although long considered a relatively minor post-translational
modification, protein citrullination (Fig. 1) has recently come to
the forefront because this modification appears to be abnormally
increased in numerous human diseases, including: multiple sclero-
sis,1 Alzheimer’s disease,2 primary open-angle glaucoma,3,4 various
adenocarcinomas,5 and rheumatoid arthritis (RA).6,7 In particular,
the links between abnormal protein citrullination and RA are espe-
cially strong and a significant body of evidence suggests that the
enzymes responsible for this modification, that is, the protein argi-
nine deiminases (PADs), are dysregulated in RA.6,7 This evidence
includes the fact that mutations within the gene encoding PAD4,
a PAD isozyme, are associated with the onset and progression of
RA in Japanese, Korean, and Caucasian populations.8–11 Additional
evidence includes the facts that the levels of PADs 2 and 4 are
increased in the RA synovium and RA patients produce autoanti-
bodies that recognize citrulline-containing proteins.12–15 The pres-
ence of these autoantibodies in patient’s sera is highly significant
because they can be detected before the onset of overt clinical
symptoms and their presence in patient’s sera is correlated with
a more severe disease course.14–16
A
H2N
HN
O
NH2
NH2
HN
PAD4
H2O
+
NH4
H
N
H
N
R
R
N
R
N
H
R
H
O
O
X
B
C
HN
NH
X = Cl, F
O
NH2
N
H
O
Mechanism 1:
The apparent causal relationship between PAD dysregulation
and RA disease onset thereby suggested these enzymes as a novel
therapeutic target for RA that could overcome the limitations of
current therapies, which focus on disease management17 rather
than the treatment of an underlying cause of the disease. Towards
that goal, we have recently described the synthesis and character-
ization of a series of haloacetamidine containing mechanism-based
inactivators that are the most potent PAD inhibitors described to
date.18,19
S Cys645
H
N
H
N
R
Cl
Cl
R
S
Cys645
NH2
NH2
Mechanism 2:
Cys645
Cys645
Cl
S Cys645
H
N
S
S
H
H
The two most potent compounds, N-a-benzoyl-N5-(2-flu-
N
N
R
L
-ornithineamide (F-amidine;kinact/KI = 3000 Mꢀ1
R
R
oro-1-iminoethyl)-
NH2
NH2
minꢀ1 19 and N-a-benzoyl-N5-(2-chloro-1-iminoethyl)-
)
L-ornithine
NH2
Figure 1. (A) PAD4 hydrolyzes peptidyl-arginine to form citrulline and ammonia.
(B) Structure of haloacetamidine-based inactivators of PAD4. (C) Two possible
mechanisms of PAD4 inactivation.
* Corresponding author. Tel.: +1 803 777 6414; fax: +1 803 777 9521.
E-mail address: thompson@mail.chem.sc.edu (P. R. Thompson).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.05.021

4384
C. P. Causey, P. R. Thompson / Tetrahedron Letters 49 (2008) 4383–4385
amide (Cl-amidine; kinact/KI = 13,000 Mꢀ1 minꢀ1),18 inactivate PAD4
by covalently modifying an active site cysteine (Cys645) that is in-
volved in nucleophilic catalysis. Although the exact mechanism of
inactivation has yet to be elucidated, it presumably proceeds
through one of two routes (Fig. 1).
The initial syntheses of F- and Cl-amidine were carried out on
the solid-phase (Rink AM amide resin) and proceeded in seven
steps with an overall yield of 70% (Scheme 1).18,19 As with all
solid-phase syntheses, this route was attractive because it miti-
gated the need to isolate intermediates by chromatography. In
addition to serving as a solid support, the Rink resin also afforded
the primary carboxamide upon acidic cleavage. While this method
does generate the desired product in reasonable yield, there are
considerable drawbacks, the most significant being the cost of
the starting materials (ꢁUS$950/g).
potent of these compounds, that is, Cl-amidine in fewer steps, with
higher yield, and at a significantly lower cost.
The solution-phase synthesis of Cl-amidine proceeded in four
steps with an overall yield of 80% (Scheme 2). This route was par-
ticularly attractive because, similarly to the solid phase synthetic
route, this procedure mitigates the need for time-consuming
intermediate chromatography because all of the intermediates
are isolated and purified by simple extractive work-ups.
The first challenge in this synthesis is differentiating between
the two amino groups of ornithine. This challenge was easily met
by choosing the mono-protected ornithine, H–Orn(Boc)–OH, a
commodity chemical that is available at a considerably lower
(ꢁ5-fold) cost than the ornithine derivative used previously. Ben-
zoylation of the a-amine was accomplished with benzoyl chloride
and afforded pure BzNH–Orn(Boc)–OH in 90% yield.20 Next, we
chose to convert the carboxylic acid into a primary amide. Using
methodology developed by Nájera and co-workers21 conversion
to the primary amide was achieved using the coupling reagent,
HOTT, with ammonium chloride.22 In the final steps, the amidine
moiety was installed on the d-amine. Removal of the Boc-group
with neat TFA proceeded in quantitative yield,23 thus enabling
the coupling of the free amine to ethyl chloroacetimidate hydro-
chloride in the presence of triethylamine.24 This coupling proce-
dure also proceeded quantitatively to afford the title compound
in a total of four steps and 80% yield.
The major cost contributor is an orthogonally protected orni-
thine (Fmoc–Orn(Dde)–OH) that must be used in large excess
(4-fold) to ensure quantitative coupling to the resin—this stoichi-
ometry means a 75% loss from the outset. A second drawback of
the solid-phase synthesis arises when the compound is cleaved
from the resin. During this step, the acidic cleavage conditions
inevitably result in some unintended hydrolysis of the primary
amide. Not only does this reduce the overall yield but it necessi-
tates the separation of the carboxylic acid and amide derivatives,
which is tedious and time-consuming. If either F- or Cl-amidine
are to become viable therapeutic candidates, a more practical syn-
thesis is necessary; and as we moved forward to mouse trials, the
need for a more cost effective method became clear. To this end,
we have designed a solution-phase strategy to synthesize the more
In conclusion, we have developed an efficient solution-phase
synthesis of Cl-amidine. Using this method, the inactivator can
be produced in fewer steps, with higher yields, and at a fraction
of the previous cost (ꢁUS$75/g).
Dde
Dde
NH
O
NH
i)
ii)
iii-iv)
H2N
FmocHN
O
H
N
H
Fmoc
N
N
N
H
H
O
v)
Cl
Cl
TFA
HN
HN
NH
O
NH
O
NH2
vii)
vi)
O
O
O
H
N
H
NH2
N
N
N
N
H
H
H
O
Scheme 1. Reagents and conditions: (i) 20% piperidine, DMF; (ii) HBTU, HOBt, 0.4 M NMM, Fmoc–Orn(Dde)–OH, DMF; (iii) 20% piperidine, DMF; (iv) BzCl, 0.4 M NMM, DMF;
(v) 2% hydrazine, DMF; (vi) ethyl chloroacetimidate (HCl), Et3N, DMF; (vii) TFA/TIS/H2O (95/2.5/2.5), 70%.
Cl
NHBoc
NHBoc
HN
NHBoc
NH
O
i)
ii)
iii-iv)
O
O
O
OH
O
OH
O
NH2
O
H2N
N
N
NH2
H
H
N
H
Scheme 2. Reagents and condition: (i) BzCl, NaOH, H2O, 90%; (ii) HOTT, DIPEA, NH4Cl, DMF, 89%; (iii) TFA, 100%; (iv) ethyl chloroacetimidate (HCl), 100%.

C. P. Causey, P. R. Thompson / Tetrahedron Letters 49 (2008) 4383–4385
17. Smolen, J. S.; Steiner, G. Nat. Rev. Drug. Discov. 2003, 2, 473–488.
4385
Acknowledgement
18. Luo, Y.; Arita, K.; Bhatia, M.; Knuckley, B.; Lee, Y. H.; Stallcup, M. R.; Thompson,
P. R. Biochemistry 2006, 45, 11727–11736.
We thank the NIH (GM079357) for financial support.
19. Luo, Y.; Knuckley, B.; Lee, Y. H.; Stallcup, M. R.; Thompson, P. R. J. Am. Chem.
Soc. 2006, 128, 1092–1093.
20. Synthesis of N-a-benzoyl-N5-(tert-butoxycarbonyl)-
carbonyl)- -ornithine (929 mg, 4.0 mmol) was dissolved in a biphasic mixture
L
-ornithine: N5-(tert-Butoxy-
Supplementary data
L
of aqueous NaOH (3.5 M, 1.16 mL, 4.0 mmol) and ether (4 mL). The mixture
was cooled to 0 °C and alternating portions of benzoyl chloride (4.0 mmol,
0.464 mL) and aqueous NaOH (8.5 M, 4.0 mmol, 0.464 mL) were added every
5 min for 30 min. The reaction mixture was allowed to warm to rt and stirred
overnight. The reaction was acidified (pH 1) with concd HCl and extracted with
dichloromethane. The organics were combined, washed with brine, dried over
MgSO4, and concentrated to afford the product as a white powder (1.2 g, 90%).
1H NMR (D2O): d 7.60 (d, 2H), 7.45–7.30 (m, 3H), 4.15 (m, 1H), 2.85 (m, 2H),
1.8–1.3 (m, 4H), 1.15 (s, 9H). 13C NMR (D2O): d 179.05, 170.34, 158.34, 133.65,
132.26, 128.87, 127.28, 80.90 55.64, 39.83, 29.17, 27.80, 25.85. HRMS
ðC17H25N2O þÞ: calcd 337.1763, observed 337.1761.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2008.05.021.
References and notes
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(3 ꢂ 12 mL), and brine (12 mL). The organics were then dried over MgSO4 and
concentrated to afford the product as a white powder (370 mg, 89%). 1H NMR
(DMSO-d6): d 8.28 (d, 1H), 7.85 (d, 2H), 7.6–7.4 (m, 3H), 7.35 (br s, 1H), 7.0 (br
s, 1H), 6.78 (br s, 1H), 4.35 (m, 1H), 2.85 (m, 2H), 1.8-1.4 (m, 4H), 1.25 (s, 9H).
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L
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(6 mL) and stirred at 0 °C for 45 min. After warming to rt, the TFA was removed
and the residue dissolved in water (5 mL) and washed with ether (5 mL) before
being lyophilized. 1H NMR (D2O): d 7.59 (m, 2H), 7.4–7.2 (m, 3H), 4.25 (m, 1H),
2.85 (m, 2H), 1.9–1.5 (m, 4H) ppm. 13C NMR (D2O): d 176.42, 171.06, 132.83,
132.63, 128.88, 127.40, 53.77, 38.99, 28.06, 23.59. HRMS ðC12H18N3O2þÞ: calcd
236.1399, observed 236.1397.
24. Synthesis of Cl-amidine: N-a-Benzoyl-L-ornithine amide–TFA (247 mg, 0.7
mmol) was dissolved in MeOH (2 mL). Triethyl amine (0.29 mL, 2.1 mmol)
and ethyl chloroacetimidate–HCl (221 mg, 1.4 mmol) were added and the
mixture stirred overnight. The reaction mixture was quenched with water
(5 mL) and acidified with TFA. The product was isolated by RP-HPLC as a white
powder. Spectral data were consistent with previous reports.