Azabenzthiazole inhibitors of leukotriene A4 hydrolase

Article abstract of DOI:10.1016/j.bmcl.2012.10.036

Previously, benzthiazole containing LTA₄H inhibitors were discovered that were potent (1-3), but were associated with the potential for a hERG liability. Utilizing medicinal chemistry first principles (e.g., introducing rigidity, lowering c Log

Full text of DOI:10.1016/j.bmcl.2012.10.036

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7504–7511
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Azabenzthiazole inhibitors of leukotriene A4 hydrolase
⇑
Virginia M. Tanis , Genesis M. Bacani, Jonathan M. Blevitt, Christa C. Chrovian, Shelby Crawford,
Aimee De Leon, Anne M. Fourie, Laurent Gomez, Cheryl A. Grice, Krystal Herman, Aaron M. Kearney,
Adrienne M. Landry-Bayle, Alice Lee-Dutra, Jay Nelson, Jason P. Riley, Alejandro Santillán Jr.,
John J. M. Wiener, Xiaohua Xue, Arlene L. Young
Janssen Research & Development LLC, 3210 Merryfield Row, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Previously, benzthiazole containing LTA4H inhibitors were discovered that were potent (1–3), but were
associated with the potential for a hERG liability. Utilizing medicinal chemistry first principles (e.g., intro-
ducing rigidity, lowering cLogD) a new benzthiazole series was designed, congeners of 1–3, which led to
compounds 7a, 7c, 12a–d which exhibited LTA4H IC50 = 3–6 nM and hERG Dofetilide Binding IC50 = 8.9–>
Received 29 August 2012
Revised 1 October 2012
Accepted 8 October 2012
Available online 17 October 2012
>10 lM.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
hERG
LogD
LTA4H
Leukotriene A4 hydrolase
Antiinflammatory
Leukotriene A4 hydrolase (LTA4H) is a zinc-containing cytosolic
enzyme with both hydrolase and aminopeptidase activity, the
crystal structure of which was published in 2001.1 LTA4H is a key
enzyme in the arachidonic acid cascade downstream of 5-lipoxyge-
nase (5-LO) and 5-lipoxygenase-activating protein (FLAP) as seen
in Fig. 1. The leukotrienes LTC4, LTD4 and LTE4, known as cysteinyl
leukotrienes,2 are also produced from LTA4.
LTA4H stereospecifically catalyzes the hydrolysis of the unstable
epoxide LTA4, affording the proinflammatory mediator leukotriene
B4 (LTB4). LTB4 is a potent chemoattractant of neutrophils, eosino-
phils, macrophages, mast cells, T-cells, dendritic cells, smooth
muscle cells and keratinocytes, and also activates neutrophils.3
This inflammatory mediator has been implicated in a plethora of
disorders, including inflammatory bowel disease (IBD),4 chronic
obstructive pulmonary disease (COPD),5 cancer,6 rheumatoid
arthritis (RA),7 asthma,8 and cardiovascular disease.9 LTA4H also
functions as an anion-dependent aminopeptidase, efficiently pro-
cessing arginyl di- and tri-peptides. Recently, a role for this amino-
peptidase activity in the degradation and inactivation of the
tripeptide proline-glycine-proline (PGP) was described.10
advantages11 over 5-LO or FLAP inhibitors which prevent the for-
mation of LTA4, a source of the lipoxins.12 In contrast, the inhibition
of LTA4H would not block the production of the lipoxins, as it acts
downstream of this branch point in the cascade. Inhibiting LTB4
formation eliminates the potential challenges associated with
antagonizing two LTB4 receptors, BLT1 and BLT2, which may be
needed in order to achieve full efficacy in vivo.13 Rao et al.11 previ-
ously reported that a LTA4H inhibitor 1 (Fig. 2) selectively blocked
LTB4 production in a zymosan-induced peritonitis model without
affecting cysteinyl leukotriene production and maintained the pro-
duction of the anti-inflammatory mediator LXA4.
In our previously disclosed report,14 the benzthiazole left hand
portion of 1 was held constant, while focusing modification efforts
on the right hand portion. These efforts resulted in a series of po-
tent and selective LTA4H inhibitors represented by 1, 2, and 3
(Fig. 2), having excellent PK/PD properties.14
The data in Table 1 show potent LTA4H inhibitory activity and
good inhibition of LTB4 production in stimulated mouse whole
blood for 1, 2, and 3.14 Compounds 1 and 2 also exhibit a relative
lack of potency inhibiting dofetilide binding to the hERG channel
(hERG binding).14 Compounds 1, 2, and 3 were evaluated in the
murine arachidonic acid-induced ear inflammation model and
were found to have good activity as measured by the % inhibition
of LTB4 production in blood, and the % inhibition of myeloperoxi-
dase activity (MPO) as a measure of neutrophil influx into the
ear tissue.15 In a rat PK study (10 mg/kg:2 mg/kg po:iv) compound
2 had a reasonable %F (65%) and t1/2 (5.6 h), and showed a Cmax of
The lipoxins, LXA4 and LXB4, derived from arachidonic acid and
LTA4, are endogenous anti-inflammatory agents that are thought to
participate in the resolution phase of inflammation (not shown).
We surmised that an inhibitor of LTA4H may have certain
⇑
Corresponding author. Tel.: +1 858 320 3332.
E-mail address: vtanis@its.jnj.com (V.M. Tanis).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.036

V. M. Tanis et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7504–7511
7505
Membrane
Inflammatory stimulus
phospholipids
COOH
Arachidonic Acid
Marketed 5-LO Inhibitor
OOH
5-LO
FLAP (5-lipoxygenase activating protein)
O
COOH
COOH
5-LO Dehydration
LTA4
5-HPETE
LTA4 Hydrolase
LTC4 Synthase
OH
OH
COOH
Cysteinyl Leukotrienes
LTB4
Figure 1. Biosynthetic pathway of the leukotrienes.
H
N
O
CO2H
N
O
N
OH
S
N
S
N
O
1
2
O
CONH2
N
S
N
3
Figure 2. Benzthiazole containing LTA4H inhibitor leads.
Table 1
In vitro and in vivo data for LTA4H inhibitors 1, 2, and 3
d,g
e
f
Compd.
LTA4H IC50 (nM)
11
13 12
17 11
MWBa LTA4H IC50 (nM)d,g
% Inhb,g LTB4/MPO
hERG DBc IC50
(
lM)
%F/ t1/2
Cmax
1
2
3
9
88 24
104 100
151 156
80/82
94/75
93/87
>10
9.9
1.5
—
—
2.2
—
65/5.6 h
—
a
b
c
d
e
f
Mouse whole blood diluted 1:15 with media is stimulated with calcium ionophore, A23187, and then assayed for LTB4 production.
Mouse arachidonic acid-induced ear inflammation model 30 mg/kg.
DB = dofetilide binding.
Data expressed as IC50 std dev in nM.
Rat PK 10 mg/kg: 2 mg/kg po:iv.
Rat PK 10 mg/kg: 2 mg/kg po:iv,
For assay details see Ref. 14.
lMol/L.
g
2.2
l
mol/L.14 However, when the potential for a hERG liability was
Some of the principles for reducing a hERG liability are reason-
ably well understood, such as introducing rigidity into flexible, rel-
atively linear molecules.16 Reducing the lipophilicity, as measured
by LogD, can be associated with a lowering of hERG interaction and
a potential reduced cardiovascular risk, but the trends are not
strong, particularly with basic compounds.17 Given these notions,
further investigated, it was found that 2 exhibited a 74% inhibition
at 3 l
M, when measured in the hERG-mediated K+ current in a
voltage clamp assay.14 Continued exploration was deemed appro-
priate in order to optimize the in vitro and in vivo activity, while
reducing a potential hERG liability.

7506
V. M. Tanis et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7504–7511
O
a
N
N
W
Cl
S
S
R
X
O
N
Y
Z
Z
W
4a-d
N
HN
R
+
S
X
15-53%
Y
Z
O
6
7a-h
W
X
CHO
HO
b
Y
5a
W
N
N
X
Y
K2CO3
Cl
+
S
CH3CN, 50°C
41-62%
Z
R
8a-c
9
a. K2CO3, CH3CN, 50°C; b. NaBH(OAc)3, ClCH2CH2Cl, RT
Scheme 1. The syntheses of thiazolo[4,5-b]pyridine, thiazolo[4,5-c]pyridine, thiazolo[5,4-c]pyridine, and thiazolo[5,4-b]pyridine congeners of 1-carbon linked analogs 1, 2
and 3 via alkylation and reductive amination.
Table 2
Compounds prepared with the chemistry of Scheme 1
Entry
Starting compd.
Product
Yield (%)
W
X
Y
Z
R
1
2
3
4
5
6
7
8
5a
4a
4b
4c
4d
8a
8b
8c
7a
7b
7c
7d
7e
7f
53
15
44
20
36
53
41
62
N
CH
N
CH
CH
N
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
N
CH
CH
C–Cl
C–F
CH
N
CONH2
CONH2
CH
CH
CH
C–CH3
CH
CH
H
H
H
H
H
H
7g
7h
N
N
Table 3
In vitro and in vivo IC50 Data for Thiazolo-Pyridines 7a–h
Compd.
LTA4H IC50 (nM)d
0.6
733 161
0.6
MWBa LTA4H IC50 (nM)d
% Inhb LTB4/MPO
hERG DBc IC50
(lM)
7a
7b
7c
7d
7e
7f
6
37 48
2043 702
14 16
723 327
ND
82 31
245 204
38 16
85/89
ND
67/48
ND
ND
12/42
ND
8.9
ND
>10
ND
ND
8.1
>10
>10
3
614 135
1800⁄
33
8
7g
7h
40 10
29 12
15/53
a–d
See Table 1 for details. (⁄) Single determination; ND = not determined.
O
N
O
N
R1
N
N
R1
N
S
S
N
O
R2
R2
10
11
O
N
R1
N
N
S
R2
12
Figure 3. Thiazolo[4,5-b]pyridine containing targets—impact of polarity/flexibility on biological activity/hERG.
follow-up efforts were initiated by examining the addition of some
polarity to the relatively lipophilic benzthiazole left hand portion
of molecules such as 1, 2, or 3.
The initial focus was on the simple paradigm of substitution
of a nitrogen into the aryl ring of the benzthiazole of 1, 2, and
3. Scheme 1 and Table 2 illustrate two construction methods.

V. M. Tanis et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7504–7511
7507
R1
R2
N
N
R1
N
O
O
N
H
N
N
Br
R2
S
S
O
O
Cs2CO3,
DMF, 50°C
6-50%
13
10a-f
Scheme 2. The synthesis of 3-atom linker containing thiazolo[4,5-b]pyridines 10.
Table 4
In vitro and in vivo IC50 data for 3-atom linker thiazolo-pyridines 10a–f
N
O
N
S
NR1R2
MWBa LTA4H IC50 (nM)d
O
Compd.
NR1R2
Yield (%)
31
LTA4H IC50 (nM)d
% Inhb LTB4 /MPO
65/62
hERG DBc IC50
>10
(lM)
10a
8
3
18
30
2
4
N
CONH2
10b
10c
50
47
16 12
80/71
89/76
>10
>10
N
N
N
O
6
7
3
3
20
6
O
N
10d
10e
6
30 20
18 10
81/86
84/72
>10
>10
N
N
26
7
4
5
2
N
O
O
HN
10f
29
24 10
85/74
>10
H
N
a–d
See Table 1 for details.
The first method utilizes either the alkylation of thiazolo-
pyridine benzylic halides 4a–d18 or the reductive amination of
the related benzaldehyde 5a,18 whereas the second method out-
lines an alternative synthetic sequence in which a more complex
right hand half 915,18 is coupled with 2-chloro-thiazolopyridines
8a–c18 to provide the target thiazolo-pyridines 7a–h. Preliminary
biological examination of 7a–h was then conducted to determine
duced ear inflammation model as measured by the inhibition of
LTB4 production and the inhibition of MPO. The addition of a sub-
stituent, such as the methyl of 7f, or the halogens of 7g or 7h offers
no obvious benefit. The lack of hERG binding associated with 7c
(IC50 > 10 lM) is noteworthy as 7c does not possess the polar car-
boxylic acid of 1 or the hydroxyacetamide of 2. As a starting point,
compound 7c exhibits a moderate cLogD value of 2.80 considering
the lack of polar or ionizable groups present in 1 and 2 (cLogD val-
ues of 1.43 and 2.58 respectively).19 These data, when taken in
concert with the in vitro and in vivo data of Table 3, clearly support
the choice of the thiazolo[4,5-b]pyridine nucleus of 7c for inclusion
in a more highly developed series of molecules.
As this study proceeded, the importance of in vitro and in vivo
assessments of biological activity (Tables 1 and 3) were considered,
while the impact of the changes upon the potential hERG liability
was examined. In order to ascertain the potential impact of a
decrease in molecular flexibility, in addition to the polarity
alterations made in Table 3, the construction of molecules with
3-, 2-, and 1-atom tethers (Fig. 3, 10–12) between the central aryl
if the pyridyl nitrogen would be tolerated, if there was
a
positional preference, and if the alteration in cLogD would
impact hERG. The data for these compounds is presented in
Table 3.
The data presented in Table 3 indicate that the thiazolo[5,4-
b]pyridine nucleus of 7b and the thiazolo[4,5-c]pyridine nucleus
of 7e are detrimental to LTA4H inhibitory activity. A direct compar-
ison of the positional isomers 7c, 7d, and 7e identifies the thiazol-
o[4,5-b]pyridine of 7c as the preferred isomer. This substitution
(7a, 7c) is associated with both potent LTA4H inhibitory activity
and good inhibition of LTB4 production in stimulated mouse whole
blood, as well as good activity in the murine arachidonic acid-in-

7508
V. M. Tanis et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7504–7511
R1
R2
O
N
O
N
N
H
R1
N
N
S
S
N
R2
OSO2CH3
Cs2CO3,
DMF, 50°C
14
11a-f
7-23%
Scheme 3. The synthesis of 2-atom linker containing thiazolo[4,5-b]pyridines 11.
Table 5
In vitro and in vivo IC50 data for 2-atom linker thiazolo-pyridines 11a–f
N
O
N
S
NR1R2
Compd.
NR1R2
Yield (%)
21
LTA4H IC50 (nM)d
MWBa LTA4H IC50 (nM)d
% Inhb LTB4 /MPO
70/36
hERG DBc IC50
>10
(lM)
11a
3
1
24 22
N
CONH2
11b
11c
23
22
11
6
3
50
9
76/60
86/75
>10
>10
N
N
N
O
14
6
65 44
22 19
O
N
11d
11e
7
8
5
86/79
90/68
>10
>10
N
N
14
3
30 12
N
O
O
HN
11f
8
0.3 0.2
43 29
85/78
8.3
H
N
a–d
See Table 1 for details.
ring and the polar right hand functionality was desired. Flexibility
will decrease as the tether length decreases and additional rigidity
can be introduced by changing the amines of 10, 11, and 12 (Fig. 3)
from simple cyclic amines to fused and bridged bicyclo[3.3.0],
bicyclo[2.2.1] and bicyclo[2.2.2] diamines.
The syntheses of a selection of 3-atom linker analogs 10, pos-
sessing the thiazolo[4,5-b]pyridine left hand ring system are
shown in Scheme 2. Bromide 1318b was treated with a variety of
amines (Cs2CO3, DMF, 50 °C) ranging from piperidine to 3-acetam-
ido-tropane18 to give amines 10a–f in modest yields (Table 4).
The target 2-atom tethered analogs were assembled in a similar
fashion, as shown in Scheme 3. The mesylate 1418b was treated
with the amines utilized in Scheme 2 (Cs2CO3, DMF) to give amines
11a–f in poor yields for the bicyclic amines (7–8%) to modest yields
for the mono-cyclic amines (21–23%; Table 5). In the case of the
bicyclic amines (11d–f, Table 5) elimination to the corresponding
styrene was found to be a major competing side reaction.
ses of these molecules, via alkylative and reductive amination pro-
tocols, are shown in Scheme 4. As expected, the utilization of the
reactive benzylic halide 4b18 as well as the reactive benzaldehyde
5a18 in this sequence afforded modest to excellent yields (30–83%)
of compounds 12a–d (Table 6), in contrast to the generally lower
yields reported in Schemes 2 and 3.
A review of the data associated with the 3-atom linker thiazolo-
pyridines 10 (Table 4) shows that 10a–f exhibit uniformly excel-
lent enzymatic inhibitory activity (IC50 of 4–16 nM) as well as
stimulated mouse whole blood activity (IC50 of 18–30 nM). The
first differentiation in this series was observed when 10a–f were
examined in vivo in the murine arachidonic acid-induced ear
inflammation model. In this assessment, the simple piperidine
10a performed poorly, inhibiting the production of LTB4 and
MPO activity to the extent of 68% and 62% respectively. The
remaining compounds of Table 4 inhibit the production of LTB4
from 80% to 89%, and inhibit MPO from 71% to 86%. This series of
molecules is the most flexible of those prepared, as a consequence
of their 3-atom tether. All of the compounds of Table 4 are inactive
The desired 1-atom tethered analogs 12 were constructed uti-
lizing only the bicyclic amines of Schemes 2 and 3, as the piperid-
inyl and piperidinyl-4-carboxamide variants had been described
earlier (Scheme 1; compounds 7c and 7a respectively). The synthe-
(IC50 > 10
lM) in the hERG binding assay, however, differentiation
of them begins in the hERG patch clamp assay.20 Compounds 10b,

V. M. Tanis et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7504–7511
7509
O
a
N
N
N
N
Cl
S
S
O
R1
N
N
R1
R2
N
4b
R2
HN
S
+
30-83%
O
12a-d
CHO
b
5a
a. K2CO3, CH3CN, 50°C; b. NaBH(OAc)3, ClCH2CH2Cl, RT.
Scheme 4. The synthesis of 1-atom linker containing thiazolo[4,5-b]pyridines 12. Ragents and conditions: (a) K2CO3, CH3CN, 50 °C; (b) NaBH(OAc)3, ClCH2CH2Cl, rt.
Table 6
In vitro and in vivo IC50 data for 1-atom linker thiazolo-pyridines 7c, 7a and 12a–d
N
O
N
S
NR1R2
Compd.
NR1R2
Yield (%)
44
LTA4H IC50 (nM)d
MWBa LTA4H IC50 (nM)d
% Inhb LTB4 /MPO
67/48
hERG DBc IC50
>10
(lM)
7c
3
6
0.6
0.6
14 16
37 48
N
CONH2
7a
53
49
85/89
81/88
8.9
N
N
N
O
12a
6
3
2
27 19
>10
O
N
12b
12c
30
53
0.6
10
7
1
57/95
92/88
>10
>10
N
N
5
1
2
1
5
N
O
O
HN
12d
83
20 17
81/70
>10
H
N
a–d
See Table 1 for details.
10e, and 10f have IC50 > 10
lM in this assay, while compounds 10c
11f shows a small hERG DB signal with an IC50 of 8.3 lM.
(IC50 = 2.5
liability.
l
M) and 10d (IC50 = 3 M) demonstrate greater hERG
l
Compounds 11c–f, were examined in a hERG patch clamp assay
and the results were most interesting.20 The 4-pyrrolidinone-
The 2-atom tether analogs (Table 5) 11a–f are associated with
excellent LTA4H inhibitory activity, with IC50 values of 0.3–
14 nM. The mouse whole blood LTA4H assessment provides a
broader range of IC50 values (22–65 nM) in this series than in the
3-atom tether 10a–f (Table 4). As was the case for the 10a–f series,
further differentiation was seen in this 2-atom tether series when
11a–f were studied in vivo in the murine arachidonic acid-induced
ear inflammation model. Compounds 11a and 11b were only
moderately active, with the in vivo assessment showing LTB4 and
MPO inhibition of <80% for each measurement. Compounds 11c–
f exhibited a better balance of LTB4 and MPO inhibitory activity.
piperidine 11c (IC50 = 2.8
were surprisingly active in this assay.
lM) and the tropane 11f (IC50 = 4.3 lM)
The final series of this study, the 1-atom tethered amines 7c, 7a,
and 12a–d were evaluated (Table 6). With the least backbone flex-
ibility of all of the molecules described herein, these compounds
provide the best opportunity to collect all of the desired attributes
in a single entity. In the in vitro LTA4H enzymatic assessment, all of
the compounds of Table 6 exhibited excellent potency (1–6 nM). A
greater range of IC50 values (7–37 nM) was observed in the mouse
whole blood LTA4H assessment, as was the case with 11a–f. In the
in vivo assessment of LTA4H target engagement, the activity of 7c
was disappointing. In contrast, the piperidinyl-4-acetamide 7a,
Compounds 11a–e exhibit IC50 > 10 lM for hERG binding, whereas

7510
V. M. Tanis et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7504–7511
Table 7
Rat pharmacokinetic data for 1-atom linker thiazolo-pyridines 12c and 12d
Compd.
Structure
IV (2 mg/kg)
Cl (L/h/kg)
PO (10 mg/kg)
t1/2 (h)
Vss (L/kg)
%F
Cmax (lM)
O
O
N
N
N
N
12c
3.3
0.42
1.2
1.4
125
3.3
1.2
S
S
N
O
O
H
HN
12d
2
3.5
65
N
N
4-pyrrolidinone 12a and the 2,5-diazabicyclo[2.2.1]heptane acet-
amide 12c are noteworthy for their good LTB4/MPO inhibitory
activities. Compound 7a inhibited dofetilide binding to the hERG
Acknowledgments
The authors are grateful to Jennifer Buenviaje for determining
the hERG IC50 values; Kia Sepassi for compound formulations and
developability; and the Bioanalytical group for PK analysis.
channel with an IC50 = 8.9
not be readily differentiated in this assay, presenting IC50 values
of >10 M. An examination of these five compounds in a hERG
patch clamp assay was also unrevealing as each is associated with
an IC50 > 10
M.20 In an attempt to discern the impact on enzy-
lM, while compounds 12a–d could
l
References and notes
l
matic inhibitory activity of alternative asymmetry (2,5-diazabicy-
clo[2.2.1]heptane acetamide) and axial vs equatorial preference
(tropane acetamide), these amines (not exemplified) were utilized
in place of those shown in Table 6 (12c and 12d) to provide com-
pounds which were identical in their in vitro and in vivo activity to
those exemplified (12c and 12d).
Compounds 7a and 12a–d (Table 6) all present a desirable col-
lection of attributes. As an example, compounds 12c and 12d were
further evaluated in rat PK (Table 7). These compounds exhibit low
to moderate clearance and a moderate to high steady state volume
of distribution. Both 12c and 12d display high oral bioavailability
and moderate intravenous half-lives.
In order to discern the impact of backbone flexibility, com-
pounds 10c (cLogD 3.28), 11c (cLogD 2.86) and 12a (cLogD
3.21) were compared. As the tether length is decreased, there is
no smooth progression in cLogD due to the electronic impact of
structural variation. However, the hERG patch clamp assay for
these compounds shows the effect of decreasing backbone
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flexibility with the 3-atom tether 10c IC50 of 2.5
2-atom tether 11c has an IC50 of 2.8 M, and the least flexible
1-atom tether 12a is associated with an IC50 > 10 M. Within the
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1.21). Five of the six compounds of Table 6 (7a, 12a–d) were exam-
ined in a hERG patch clamp assay and all compounds had
lM, while the
l
l
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the tether and introducing amines with additional polarity and/or
rigid cage structures, while keeping within a relatively narrow
range of cLogD, has a positive effect on the hERG patch clamp
outcome.
The data of Table 6 suggest that the design plans to continue to
optimize the desirable LTA4H activity while minimizing potential
liabilities has been realized. Reducing flexibility appears to have
greatly reduced the potential for a hERG liability, as measured in a
patch clamp assay. This stands in contrast to the more standard
hERG binding assessment, which for this series did not allow for
sufficient prioritization. The 1-atom linker molecules may offer
the best combination of properties to warrant further investigation.

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19. Calculated with ACD Labs Software v12.0.
20. Determined by AVIVA using PatchXpress™.