Structure and property based design of factor Xa inhibitors: pyrrolidin-2-ones with monoaryl P4 motifs

Article abstract of DOI:10.1016/j.bmcl.2009.11.077

Structure and property based drug design was exploited in the synthesis of sulfonamidopyrrolidin-2-one-based factor Xa inhibitors, incorporating neutral and basic monoaryl P4 groups, ultimately producing potent inhibitors with effective levels of anticoagulant activity and extended oral pharmacokinetic profiles. However, time dependant inhibition of Cytochrome P450 3A4 was a particular issue with this series.

Full text of DOI:10.1016/j.bmcl.2009.11.077

Bioorganic & Medicinal Chemistry Letters 20 (2010) 618–622
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure and property based design of factor Xa inhibitors:
pyrrolidin-2-ones with monoaryl P4 motifs
Savvas Kleanthous a, Alan D. Borthwick a, David Brown a, Cynthia L. Burns-Kurtis b, Matthew Campbell a,
Laiq Chaudry a, Chuen Chan a, Marie-Olive Clarte a, Máire A. Convery a, John D. Harling a, Eric Hortense a,
Wendy R. Irving a, Stephanie Irvine a, Anthony J. Pateman a, Angela N. Patikis a, Ivan L. Pinto a,
Derek R. Pollard a, Theresa J. Roethka b, Stefan Senger a, Gita P. Shah a, Gary J. Stelman b, John R. Toomey b,
Nigel S. Watson a, Robert I. West a, Caroline Whittaker a, Ping Zhou a, Robert J. Young a,
*
a GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
b GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure and property based drug design was exploited in the synthesis of sulfonamidopyrrolidin-2-
one-based factor Xa inhibitors, incorporating neutral and basic monoaryl P4 groups, ultimately
producing potent inhibitors with effective levels of anticoagulant activity and extended oral pharma-
cokinetic profiles. However, time dependant inhibition of Cytochrome P450 3A4 was a particular issue
with this series.
Received 18 September 2009
Revised 16 November 2009
Accepted 17 November 2009
Available online 20 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Factor Xa
Oral inhibitors
Property based design
Structure based design
P450 inhibition
The selective inhibition of proteases in the blood coagulation
cascade has been extensively researched in the pursuit of new
small-molecule anticoagulant therapies.1 This approach offers the
prospect of improved efficacy in suppressing thrombosis, whilst
reducing the bleeding liability associated with other therapies,
thus eliminating the need for intensive monitoring. Recent approv-
als for selective oral therapies have been achieved with the throm-
bin inhibitor dabigatran2 and the factor Xa (fXa) inhibitor
rivaroxaban,3 whilst other molecules have progressed to clinical
studies.4
Our programme of work on fXa inhibitors has focussed on the
sulfonamidopyrrolidin-2-one template, exemplified by our first
clinical candidate 1,5 featuring the morpholine alanyl amide P4
ligand. We successfully exploited structure based design to explore
alternative ligands within this pocket as the basis of our back-up
programme, which produced many molecules with attractive
levels of potency, but at the cost of sub-optimal overall profiles.
Notably, the biaryl derivatives 2 and 3 displayed encouraging phar-
macokinetic (PK) exposure,6 but their excellent potency translated
into relatively modest anticoagulant activity (Table 1).
Compounds made to this point were designed within strict
hydrophobicity limits (c log D7.4 <4)9, which often showed tangi-
ble benefits in terms of better translation of activity into antico-
agulant activity and improved pharmacokinetics. Furthermore, a
review of PK data alongside structural properties indicated that
a reduction in molecular size was a potential means of improving
***
exposure.
In this communication, these property based design
principles are highlighted in the discovery of a new series of
inhibitors.
Inspection of the X-ray crystal structure of 2 bound into fXa
suggested that its distal ring could be truncated to either an isopro-
penyl or carboxamide group, whilst maintaining hydrophobic con-
tacts with the aromatic resides Tyr99, Trp215 and Phe174 in the S4
pocket (Fig. 1). This assessment provided impetus to investigate a
novel series of compounds that offered opportunities, through loss
of an aromatic ring,10 to significantly reduce size and maintain or
lower hydrophobicity levels.
Synthetic entry into these compounds was established
through tert-butyl (3S)-1-(2-fluoro-4-iodophenyl)-2-oxopyrroli-
din-3-yl carbamate 4, akin to the 4-bromo derivative described
in the biaryl series6a (Scheme 1). The 2-propenyl substituted
* Corresponding author. Tel.: +44 1438 768372.
E-mail address: Rob.J.Young@gsk.com (R.J. Young).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.077

S. Kleanthous et al. / Bioorg. Med. Chem. Lett. 20 (2010) 618–622
619
Table 1
ble 2); with amide 10 being of similar size (expressed as calcu-
lated molar refractivity = 10.7),9 molecular weight (444) and
hydrophobicity (c log D7.4 2.3) to our first clinical candidate 1
(Table 1).
To capitalise on these findings, our previously successful tactic
of improving physical properties by introducing a weakly basic
centre into the motif binding in the S4 pocket was explored.6b,11
This was best achieved, whilst maintaining a similar size and
topology, by formal replacement of the carbonyl oxygen of 10
fXa inhibitory activities,7 anticoagulant potency8 and physical descriptors9 for
compounds 1–3
H
S
N
O S
O
Cl
N
O
R
R
O
N
O
1
H
N
O S
by an alkyl group, thereby generating an
lamine.
a-methyl benzy-
N
H
N
O S
R
O
N
Racemic and chiral approaches were exploited in the synthesis
of the target compounds. Thus acetophenone 13 was converted
into the racemic aminoethyl anilines 14 by reductive amination
followed by acidic deprotection of the Boc-protecting group
(Scheme 2). Chiral analogues were accessed by reduction of the
imine derived from TiCl4 mediated condensation of 13 with either
(S)- or (R)-1-phenylethylamine12 to give 15 or its enantiomer; this
proceeded with high diastereoselectivity.13 Subsequently, Eschwe-
iler–Clarke methylation, hydrogenolysis of the chiral auxiliary and
Boc removal/selective re-introduction were performed in an
appropriate order to furnish anilines 16 or 17 of high enantiopurity
for further manipulation (Scheme 2).
O
F
MeO2S
N
R
O
O
N
F
Me2N
3
2
Entry
fXa Ki (nM)
1.5 ꢀ PT (lM)
c log D7.4
MW
cmr
1
2
3
4
0.2
0.2
1.2
21.3
4.2
2.1
3.0
2.7
447
555
524
10.9
13.7
13.3
intermediate 5 was prepared via the palladium mediated cou-
pling of 4 with the organozinc reagent generated in situ from
2-bromo-1-propene. Modified Heck coupling of 4 with butyl vinyl
ether followed by acid hydrolysis of the derived (1:1) mixture of
Protected amino pyrrolidin-2-ones 18 were constructed
(Scheme 3) using homoserine lactone/trimethyl aluminium meth-
odology14 when an unprotected benzylamine was present; alterna-
tively, the orthogonally protected intermediates 19 were accessed
from N-Cbz-methionine.15 These were converted into the target
compounds 20–35 using the established sequences with appropri-
ate manipulation of Cbz and Boc-protecting groups around the
sulfonylation step.
The high potency generated in this series is first illustrated
in Table 3 for the N,N-dimethylamino analogues, with the (E)-
2-(5-chlorothien-2-yl)ethenyl P1 motif. The initial compound
20, as a mixture of two diastereomers, showed both excellent
inhibition of fXa and good anticoagulant activity; furthermore,
the individual diastereomers (21 and 22) showed closely similar
profiles.
a
and b substituted vinyl ethers afforded methylketone 6. Pri-
mary 7a, secondary 7b, and tertiary 7c amide intermediates were
prepared via palladium mediated carbonylation of 4 followed by
trapping with the ammonia, methylamine or dimethylamine,
respectively. Each intermediate was deprotected under acidic
conditions furnishing amines that were derivatised as the (E)-2-
(5-chloro-2-thienyl)ethenesulfonamides to yield target com-
pounds 8–12.
Gratifyingly, this set of compounds displayed low nM levels
of fXa inhibition7 and, for the more hydrophilic examples,
encouraging levels of anticoagulation8 (expressed as 1.5 ꢀ PT, Ta-
With this basic P4 moiety, the slightly bulkier 6-chloronaphthyl
P1 group was also accommodated (25 and 26), whilst maintaining
good overall physical properties. Although resulting in slightly
reduced anticoagulant activity, this modification had beneficial
effects on PK profiles, with 25 and 26 displaying low clearances
and increased volumes of distribution that contributed to
improved half-lives. A range of alkyl substituents on the pendant
amine group was investigated (27–33, Table 4), but none of the
derived analogues showed improved in vitro potency when
compared with the N,N-dimethyl analogues. However, it is
noteworthy that better translation of enzyme inhibitory activity
into anticoagulant activity was seen across the series when com-
pared with that observed in the biaryl series exemplified by 2
and 3.
In vitro metabolism studies indicated rapid removal of one
methyl group from the N,N-dimethyl analogues 25 and 26, giv-
ing the N-monomethyl analogues 34 and 35, which were inde-
pendently synthesised and evaluated (Table 5). Whilst these
compounds showed appreciably poorer anticoagulant activities
than 25 and 26, the significantly extended half–lives were indic-
ative of less-frequent dosing regimes. However, the progression
of this series was halted due to strong time dependant inhibition
of Cyp3A4,17 which was attributed to the benzylic amine
sub-structure, as it was independent of the P1 substituent.
Extensive metabolism of a benzylamine group was noted in
DPC423,18 another fXa inhibitor, which gave rise to reactive
intermediates that could cause irreversible damage of the Cyp
structure.19
Figure 1. Overlay of a proposed carboxamide onto the X-ray crystal structure of 2
bound into fXa (PDB code 2vh6)6a, showing the contacts in the S4 pocket.

620
S. Kleanthous et al. / Bioorg. Med. Chem. Lett. 20 (2010) 618–622
F
F
a
N
I
N
O
NHBoc
O
NHBoc
e, f
5
4
b, c
F
d
O
O
F
N
O
S
O
N
H
F
N
O
N
NHBoc
S
N
O
8
O
R1
6
NHBoc
Cl
R2
7
e, f
e, f
F
F
O
O
N
O
O
O
O
N
N
O
S
R1
S
N
H
N
H
R2
O
S
9
S
10 R1 = R2 = H,
11 R1 = Me, R2 =H
12 R1 =R2 = Me
Cl
Cl
Scheme 1. Reagents and conditions: (a) nBuLi, MeC(Br)CH2, THF, ꢁ78 °C; then ZnCl2, Et2O; PdCl2(PPh3)2, THF, ꢁ78–20 °C; (b) butyl vinyl ether, Na2CO3, Et3N, Pd(OAc)2, DPPP,
DMF, 80 °C; (c) 0.1% HCO2H, H2O, MeCN, rt; (d) CO (gas), PdCl2(PPh3)2 DMF, NH3, MeNH2 or Me2NH, 80 °C; (e) 4 M HCl/Dioxane, rt; (f) RSO2Cl, DIPEA, MeCN, rt.
Table 2
fXa inhibitory activities7 anticoagulant potency8 and physical descriptors9 for
compounds 8–12
NH2
NHBoc
NH2
F
F
F
a, b
F
16
Me
14
R2
Cl
13
S
X
N
O
O
S
N
O
N
N
H
R1
Me
O
c
(or enantiomer)
NHBoc
d, e, b
NH2
F
F
Entry
X
fXa Ki (nM) 1.5 ꢀ PT (lM) c log D7.4 cmr
MW
e, d, b, f
15
(S,S)
8
9
10
11
12
C(CH2)CH3
C(O)CH3
C(O)NH2
C(O)NHMe 35
C(O)NMe2 1.5
6.2
6.0
5.1
>100
>100
7.7
19.6
6.5
3.9
3.2
2.3
2.3
2.0
11.2 441
10.8 443
10.7 444
11.1 458
11.6 472
17
Boc
N
N
H
Ph
Me
(or enantiomer)
(or enantiomer)
Scheme 2. Reagents and conditions: (a) R1R2NH, NaHB(OAc)3, AcOH, THF, rt; (b)
HCl, dioxane, rt; or HCl, MeOH, rt; (c) PhCH(Me)NH2, Et3N, TiCl4, PhMe, ꢁ78 °C; then
NaBH4, MeOH ꢁ78 °C; (d) H2, 50 psi, Pd(OH)2–C, EtOH 60 °C; (e) H2C@O, HCO2H,
Although the benzylic amine motif in this series and that in
DPC423 are closely similar, this is not reflected in their fXa binding
modes. The 3-aminomethylphenyl group in DPC423 binds in the
P1 pocket20 whereas X-ray structures21 of 21 and 22 bound into
fXa (Fig. 2) clearly showed the related sub-unit in the P4 pocket
where the diastereomeric methyl groups make hydrophobic con-
tacts with the Trp215 residue and the amino group nitrogen is
within hydrogen bonding distance of the backbone carbonyl of
Glu97. The S1 pocket is occupied by the aromatic sulfonamide
group as seen throughout previously reported series.5,6,11
MeOH,
D; (f) Boc2O, DCM, rt.
ilar to our first clinical candidate, resulting in effective anticoagu-
lant activity with enhanced oral pharmacokinetic half–lives.
However, further profiling of the molecules indicated time depen-
dant inhibition of Cyp3A4. Important lessons learned with these
compounds were subsequently exploited in potent, long-acting
molecules without P450 liabilities, which will be reported in future
communications.
In conclusion: the compounds described in this paper further
explored chemical space, defined by size and hydrophobicity, sim-

S. Kleanthous et al. / Bioorg. Med. Chem. Lett. 20 (2010) 618–622
NHBoc
621
O
S
O
N
H
N
b
a +
NH2
R1
N
R3
O
O
e, g
NHBoc
R2
F
O
18
F
O
N
when R1 = R2 = alkyl
c,d
F
R2
N
R1
R2
1'
N
N
R1
20 - 35
f, g, e
when R1 = Me, R2 = Boc
Me
N
NHCbz
Boc
F
O
(or 1'-epimer)
19
Scheme 3. Reagents and conditions: (a) Me3Al, DCM, 0 °C to rt; (b) DtBAD, Bu3P, THF, rt; (c) CbzMetOH, HATU, DIPEA, DCM, rt; (d) MeI, MeCN, 60 °C, then Cs2CO3; (e) HCl,
MeOH, rt; (f) H2, Pd(OH)2–C, EtOH, rt; (g) R3SO2Cl, pyridine, MeCN, rt.
Table 3
fXa inhibitory activities,7 anticoagulant potency,8 physical descriptors9 and PK parameters16 for compounds 20–26
3S
H
N
N
S
O
R3
O
O
1'
Me
F
NMe2
R3
fXa Ki (nM)
1.5 ꢀ PT (
l
M)
MW
472
c log D7.4
cmr
12.0
Clpa (ml/min/kg)
T1/2 (h)
Vssc (L/kg)
Fd (%)
b
Entry
10-Stereo
20
21
22
R/S
R
S
0.8
2
1
0.9
0.7
0.9
—
7.5
4.3
—
—
0.66
0.53
—
54
23
2.2
1.3
1.6
Cl
S
23
R/S
5
3.8
496
490
2.4
2.4
12.8
12.9
2.1
3.1
0.52
10
S
Cl
Cl
24
25
R/S
R
0.8
2
2.9
1.9
-
8
—
2.6
—
1.3
—
43
26
S
2
2.1
13
2.1
1.8
81
a
b
c
Clp, plasma clearance expressed as mL/min/kg.
T1/2, half-life of the test compound expressed in hours.
Vss, steady state volume of distribution expressed as L/kg.
F, oral bioavailability expressed as percentage.
d
Table 4
fXa inhibitory activities7 and anticoagulant potency8 for compounds 27–33
Table 5
fXa inhibitory activities,7 anticoagulant potency,8 physical descriptors9 and PK
parameters16 for compounds 34 and 35
Cl
F
H
N
N
3S
S
O
O
F
Cl
Me
R1
O
H
N
S
1'R
Me
N
Ar
N
1'S
R2
Me
O
O
NHMe
O
34
35
NHMe
Entry
NR1R2
fXa Ki (nM)
1.5 ꢀ PT (lM)
24
27
28
29
30
31
32
33
NMe2
NEtMe
NHEt
0.8
6
3
20
9
4
2.9
4.4
11.2
23.6
6
5.3
4.1
5.5
b
Entry 10-
fXa
1.5
MW c log cmr Clpa
T1/2 Vssc
Fd
Stereo Ki (nM) ꢀ PT
D7.4
(ml/
(h) (L/kg) (%)
(l
M)
min/kg)
NH-i-Pr
34
35
R
S
4
2
6.9
7.0
476 1.6
476 1.6
12.5 1.6
12.5 3.1
18
15
2.4
3.3
>10A
>15A
NMe-i-Pr
Azetidinyl
Pyrrolidinyl
Piperidinyl
4
2
a,b,c,d
as Table 3.
A
These F% data are low estimates of the true figure due to the extended half-lives.

622
S. Kleanthous et al. / Bioorg. Med. Chem. Lett. 20 (2010) 618–622
7. Factor Xa inhibitory activities were determined using Rhodamine 110, bis-
(CBZ-glycylglycyl)-
-arginine amide as the fluorogenic substrate; details are
described in Ref. 5a.
L
8. Anticoagulant activities were determined in the prothrombin time (PT) assay;
see Ref. 5a, expressed as the concentration required to extend the control
coagulation time by 50% (1.5 ꢀ PT).
9. Hydrophobicity predictions, expressed as c log D7.4, were all re-calculated using
Advanced Chemistry Development software v11.0 to ensure consistency in this
paper; calculated molecular refractivity (cmr) was derived from Daylight
software v4.9.
10. Ritchie, T. J.; MacDonald, S. J. F. Drug Discovery Today 2009, 14, 1011.
11. Young, R. J.; Campbell, M.; Borthwick, A. D.; Brown, D.; Burns-Kurtis, C. L.;
Chan, C.; Convery, M. A.; Crowe, M. C.; Dayal, S.; Diallo, H.; Kelly, H. A.; King, N.
P.; Kleanthous, S.; Mason, A. M.; Mordaunt, J. E.; Patel, C.; Pateman, A. J.;
Senger, S.; Shah, G. P.; Smith, P. W.; Watson, N. S.; Weston, H. E.; Zhou, P.
Bioorg. Med. Chem. Lett. 2006, 16, 5953.
12. (a) Wu, Y.-J.; He, H.; Sun, L.-Q.; Wu, D.; Gao, Q.; Li, H.-L. Bioorg. Med. Chem. Lett.
2003, 13, 1725; (b) Nugent, T. C.; Ghosh, A. K.; Wakchaure, V. N.; Mohanty, R. R.
Adv. Synth. Catal. 2006, 348, 1289.
13. The reduction to produce 15 typically proceeded with about 9:1
diastereoselectivity. This diastereomeric ratio was enhanced by normal phase
chromatography, typically furnishing product of >90% ee at this centre.
14. Bell, I. M.; Beshore, D. C.; Gallicchio, S. N.; Williams, T. M. Tetrahedron Lett.
2000, 41, 1141. Typically this reaction furnished products with ee’s comparable
to those reported in this paper and Ref. 6, as determined by 19F NMR of Mosher
amide derivatives of deblocked 18 and/or by chiral hplc analysis of final
compounds.
Figure 2. X-ray crystal structures of the compounds 21 and 22 bound into fXa,
highlighting S4 interactions as described in the text.
15. See Ref. 5a; 19F NMR of Mosher amides of the intermediate amines formed by
hydrogenolysis of 19 suggested these transformations gave homochirality at
this centre.
References and notes
16. Pharmacokinetics measured in male Sprague–Dawley rats following
intravenous and oral administration. The formulation used for both iv and po
dosing was a 5:95% (v/v) mixture of DMSO and 50:50 PEG-200: sterile water.
Serial blood samples were collected into heparinised containers at various
time-points and blood centrifuged to yield plasma. These studies used at least
three animals for each (iv/po) leg.
1. (a) Gustafsson, D.; Bylund, R.; Antonsson, T.; Nilsson, I.; Nystroem, J.-E.;
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(b) Saiah, E.; Soares, C. Curr. Top. Med. Chem. 2005, 5, 1677.
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Plosker, G. L. Drugs 2008, 68, 1699; (c) Sobera, L. A.; Bozzo, J.; Castaner, J. Drugs
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Chaudry, L.; Chung, C.-W.; Convery, M. A.; Hamblin, J. N.; Johnstone, L.; Kelly, H.
A.; Kleanthous, S.; Patikis, A.; Patel, C.; Pateman, A. J.; Senger, S.; Shah, G. P.;
Toomey, J. R.; Watson, N. S.; Weston, H. E.; Whitworth, C.; Young, R. J.; Zhou, P.
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Valocik, R. E.; Koster, P. F.; Amour, A. J.; Chan, C.; Brown, D.; Chaudry, L.; Zhou,
P.; Patikis, A.; Patel, C.; Pateman, A. J.; Young, R. J.; Watson, N. S.; Toomey, J. R. J.
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6. (a) Young, R. J.; Borthwick, A. D.; Brown, D.; Burns-Kurtis, C. L.; Campbell, M.;
Chan, C.; Charbaut, M.; Chung, C.-w.; Convery, M. A.; Kelly, H. A.; King, N. P.;
Kleanthous, S.; Mason, A. M.; Pateman, A. J.; Patikis, A. N.; Pinto, I. L.; Pollard, D.
R.; Senger, S.; Shah, G. P.; Toomey, J. R.; Watson, N. S.; Weston, H. E. Bioorg. Med.
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Kurtis, C. L.; Campbell, M.; Chan, C.; Charbaut, M.; Convery, M. A.; Diallo, H.;
Hortense, E.; Irving, W. R.; Kelly, H. A.; King, N. P.; Kleanthous, S.; Mason, A. M.;
Pateman, A. J.; Patikis, A. N.; Pinto, I. L.; Pollard, D. R.; Senger, S.; Shah, G. P.;
Toomey, J. R.; Watson, N. S.; Weston, H. E.; Zhou, P. Bioorg. Med. Chem. Lett.
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17. Time dependant inhibition was determined by comparing the activity of
CYP3A4 in bactosomes (Cypex ltd, Dundee, Scotland) when incubated with
compound at its IC50 for zero and 25 min (10
lM concentration of 34 and
33 of 35). Over 25 min 34 showed 17-fold decrease in activity of
l
M
a
Diethoxyfluorescein (DEF) substrate turnover with Cyp3A4 and 35 an 8.5-fold
decrease. Experiments with human liver microsomes gave similar results.
18. (a) Mutlib, A. E.; Shockcor, J.; Chen, S.-Y.; Espina, R. J.; Pinto, D. J.; Orwat, M. J.;
Prakash, S. R.; Gan, L.-S. Chem. Res. Toxicol. 2002, 15, 48; (b) Mutlib, A. E.; Chen,
S.-Y.; Espina, R. J.; Shockcor, J.; Prakash, S. R.; Gan, L.-S. Chem. Res. Toxicol. 2002,
15, 63.
19. (a) Kalgutkar, A. S.; Gardner, I.; Obach, R. S.; Shaffer, C. L.; Callegari, E.; Henne,
K. R.; Mutlib, A. E.; Dalvie, D. K.; Lee, J. S.; Nakai, Y.; O’Donnell, J. P.; Boer, J.;
Harriman, S. P. Curr. Drug Metab. 2005, 6, 161; (b) Kalgutkar, A. S.; Obach, R. S.;
Maurer, T. S. Curr. Drug Metab. 2007, 8, 407.
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Cacciola, J.; Rossi, K. A.; Alexander, R. S.; Smallwood, A. M.; Luettgen, J. M.;
Liang, L.; Aungst, B. J.; Wright, M. R.; Knabb, R. M.; Wong, P. C.; Wexler, R. R.;
Lam, P. Y. S. J. Med. Chem. 2001, 44, 566.
21. The structure for 21 was refined at 1.9 Å (overall Rmerge is 0.089) in Refmac5
to a final Rfactor of 0.196 and Rfree of 0.234 and 22 was refined at 2.4 Å (overall
Rmerge is 0.055) in Refmac5 to a final Rfactor of 0.197 and Rfree of 0.255 using
procedures described in Ref. 5a. Coordinates are deposited in the protein data
bank with codes 2wyg 21 and 2wyj 22.