Lead optimization of the VU0486321 series of mGlu1 PAMs. Part 3. Engineering plasma stability by discovery and optimization of isoindolinone analogs

Article abstract of DOI:10.1016/j.bmcl.2016.03.031

This Letter describes the further lead optimization of the VU0486321 series of mGlu₁ positive allosteric modulators (PAMs), focused on addressing the recurrent issue of plasma instability of the phthalimide moiety. Here, we evaluated a number of phthalimide bioisosteres, and ultimately identified isoindolinones as the ideal replacement that effectively address plasma instability, while maintaining acceptable mGlu₁ PAM potency, DMPK profile, CNS penetration and mGluR selectivity.

Full text of DOI:10.1016/j.bmcl.2016.03.031

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1869–1872
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lead optimization of the VU0486321 series of mGlu1 PAMs. Part 3.
Engineering plasma stability by discovery and optimization of
isoindolinone analogs
Pedro M. Garcia-Barrantes a, Hyekyung P. Cho a,b, Anna L. Blobaum a, Colleen M. Niswender a,b
,
P. Jeffrey Conn a,b, Craig W. Lindsley a,b,c,
⇑
a Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
c Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter describes the further lead optimization of the VU0486321 series of mGlu1 positive allosteric
modulators (PAMs), focused on addressing the recurrent issue of plasma instability of the phthalimide
moiety. Here, we evaluated a number of phthalimide bioisosteres, and ultimately identified isoin-
dolinones as the ideal replacement that effectively address plasma instability, while maintaining accept-
able mGlu1 PAM potency, DMPK profile, CNS penetration and mGluR selectivity.
Received 15 February 2016
Revised 8 March 2016
Accepted 9 March 2016
Available online 10 March 2016
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
mGlu1
Metabotropic glutamate receptor
Positive allosteric modulator (PAM)
Schizophrenia
Structure–activity relationship (SAR)
Recent genetic data implicating GRM1 in schizophrenia1–3 and
studies showing that the adverse affect liabilities of group I meta-
botropic glutamate receptors (mGluRs) is mediated by mGlu5 and
not mGlu1,4 have rekindled interest in the development of mGlu1
positive allosteric modulators (PAMs).3–6 However, early mGlu1
PAMs lacked the DMPK profiles to serve as robust in vivo tool com-
pounds (Fig. 1).7
In response, our lab has focused efforts on developing novel
mGlu1 PAMs,3–6 with an intent to discover the ideal in vivo tool
compound to then validate the target for multiple neuropsychiatric
disorders. Within the phthalimide-based VU0486321 series of
mGlu1 PAMs, most optimization parameters could be effectively
addressed (potency, disposition, CNS penetration), except for
reocurring and variable in vitro plasma instability due to hydroly-
sis of the phthalimide moiety.3–6,8 In this Letter, we explore phthal-
imide biosiosteres and other replacements, ultimately identifying
isoindolinones as the ideal substitute, and the discovery of
VU0487351, a potent, selective, CNS penetrant and stable mGlu1
PAM tool compound.
moieties to provide isoindolionones and isoindolines (Fig. 2) to
deliver diverse analogs 6. The chemistry required is straight
forward, as shown in Scheme 1, requiring only three steps from
commercial materials to access final compounds 9, 13 and 16.
Nucleophilic aromatic substitution between 7 and different amines
produced intermediates 8, which where hydrogenated and submit-
ted to amide coupling with 3-methyl-2-furoic acid to give analogs
9. Functionalized p-amino nitroarenes/heteroarenes 10 were
condensed with various anhydrides to afford analogs 11. The nitro
group was reduced to the aniline 12 via hydrogenation conditions,
and final analogs 6 were afforded by standard amide coupling
conditions with a diverse array of heterocyclic carboxylic acids to
provide analogs 13. Alternatively, 7 could be employed to displace
benzylic bromides 14, which upon heating formed the c-lactams
15. Then, nitro reduction and acylation would yield the diversely
functionalized isoindolinones 16.9
Initially, we evaluated analogs of 3, and the SAR was intriguing.
Expansion of the phthalimide moiety to an isoquinoline dione 17
(Fig. 2) afforded
a
potent mGlu1 PAM (EC50 = 242 nM,
The optimization plan targeted expansion of the phthalimide
moiety, saturated analogs and sequential deletion of carbonyl
pEC50 = 6.65 0.09, 105% Glu Max)10 with a clean CYP profile, but
high intrinsic clearance (13.5 mL/min/kg and 54 mL/min/kg for
human and rat, respectively) and was found to be unstable in both
rat and human plasma. Further expansion to the seven-membered
benzo[d]azepine core 18, led to a significant loss of activity
⇑
Corresponding author.
E-mail address: craig.lindsley@vanderbilt.edu (C.W. Lindsley).
http://dx.doi.org/10.1016/j.bmcl.2016.03.031
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

1870
P. M. Garcia-Barrantes et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1869–1872
Cl
F
O
O
Cl
O
O
Cl
N
O
N
NH
H
N
O
N
O
N
F3C
N
H
O
O
O
Cl
3, VU0486321
2,
VU0483605
1
, Ro 07-11401
hmGlu1 EC50 = 31.8 nM (98%)
improved PK, Kp, plasma stability
~35-fold selective vs. mGlu4
hmGlu1 PAM (EC50 = 390 nM)
>10 µM versus mGlu4
O
F O
O
O
F
O
O
N
NH
N
NH
O
O
4, VU6002194
hmGlu1 EC50 = 12.9 nM (84%)
>793-fold selective vs. mGlu4
5, VU6004909
hmGlu1 EC50 = 25.7 nM (70%)
>450-fold selective vs. mGlu4
Figure 1. Structures of representative mGlu1 PAMs 1–5.
(EC50 = 3.5
3,4-dihydroquinolinone congener 19, was a modest mGlu1 PAM
(EC50 = 1.29 M, pEC50 = 5.88 0.09, 107% Glu Max) with suppra-
hepatic clearance; however, 19 was stable in human and rat
plasma, and suggested lactams may be productive in engendering
plasma stability (Fig. 3).
lM, pEC50 = 5.44 0.08, 95% Glu Max). Interestingly, a
delete
R2
F
iterative
parallel
O
Cl
O
R1
O
O
N
O
O
l
Het
N
NH
NH
synthesis
n
delete
O
Based on these data, we then explored a diverse array of phthal-
imide replacements 20, with varying degrees of success (Table 1).
Representative examples include 20a and 20b, the direct, saturated
(both cis- and trans-isomers) of 3, which proved to be inactive. The
hydrolyzed product of 3, 20c, was synthesized and found to be
active (EC50 = 930 nM, pEC50 = 6.03 0.14, 108% Glu Max); how-
ever, 20c was not CNS penetrant and displayed poor disposition,
yet an ‘active’ in vitro metabolite. Other benzoates, with diverse
functional groups in place of the carboxylic acid were all inactive.
Isoindolinone 20e was active, but the regioisomeric congener 20g
was inactive, and both regioisomeric isoindolines, 20f and 20h
were active. 20l, a regiosiomer of 19 was potent (EC50 = 780 nM,
pEC50 = 6.11 0.12, 94% Glu Max) as well. Profiling of all the active
analogs 20 in our in vitro DMPK assays quickly led us to focus on
5, VU0486321
6
Figure 2. Chemical optimization plan to replace the phthalimide moiety of 5 with
novel analogs 6.
R1
NH
Cl
Cl
Cl
O
O
R2
a
R1
b,c
R1
N
NO2
F
NO2
N
NH
R2
R2
7
8
9
R1
R1
O
N
the isoindolinone 17e (EC50 = 3.72 lM, pEC50 = 5.43 0.19, 91%
O
O
N
R2
b,c
R2
Het
Glu Max), as it displayed complete stability in rat and human liver
microsomes with low intrinsic clearance. Now, the focus was to
improve mGlu1 PAM potency.
NO2
NH
15
16
For the next iteration of parallel synthesis, we surveyed func-
tionalized isoindolinone congeners 21, of the mGlu1 PAM 3
(Table 2). Gratifyingly, substituents on the isoindolinone phenyl
ring increased potency by >10-fold in some cases, providing mGlu1
PAMs with nanomolar potency.
Evaluation of analogs 21 in our in vitro and in vivo battery of
DMPK assays quickly identified 21a (VU0487351),3–6,11 as an
exceptional compound. First, and in contrast to 3–5, 21a was
hydrolytically stable in rat and human liver microsomes and dis-
played moderate intrinsic clearance (rat CLhep 54.1 mL/min/kg
and human CLhep 8.53 mL/min/kg). Moreover, 21a had a clean
O
R2
OMe
Br
d
14
R1
O
N
O
R1
R2
d
b
NO2
H2N
NO2
n
10
11
R1
CYP profile (IC50s > 30
inactive at mGlu4 (EC50 > 10
l
M against 3A4, 1A2, 2C9 and 2D6), was
M) and was found to be highly CNS
O
N
O
R2
R1
O
N
O
O
R2
l
c
Het
NH2
penetrant (Kp = 1.36, Cn plasma 193 nM and Cn brain 269 nM). In
vivo, 21a displayed favorable rat PK, with a low clearance (CLp
11.1 mL/min/kg), high distribution volume (VD 3.36 L/kg) and good
half-life (t1/2 = 93 min, MRT = 302 min). Therefore, the isoin-
dolinone moiety solved the plasma instability issue, as well as
affording a favorable in vitro and in vivo DMPK profile. The only
blemish for 21a was high protein binding (>99% in human and
NH
n
n
12
13
Scheme 1. Reagents and conditions: (a) K2CO3, ACN, 60 °C, 43–89%, (b) H2, Pd/C,
EtOH, rt, 94–99%; (c) heterocyclic carboxylic acids, HATU, DCM, rt, 39–98%; (d) aryl
anhydrides, AcOH, reflux, 64–94%; (e) K2CO3, DMF, lW, 150 °C, 15 min, 29–92%.

P. M. Garcia-Barrantes et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1869–1872
1871
O Cl
N
O
O
O Cl
N
O
Cl
O
O
O
NH
NH
N
O
NH
O
O
17
18
19
Figure 3. Homologated analogs 14–16 of 3 that retained mGlu1 PAM activity.
Table 1
Table 2
Structures and activities for analogs 20
Structures and activities for analogs 21
Cl
R
O
O
O
7
Cl
O
O
NH
R
N
NH
20
4
21
hmGlu1 EC50
Cpd
Het
hmGlu1 EC50
[% Glu Max SEM]
(l
M)a
mGlu1 pEC50
( SEM)
Cpd
R
(l
M)a
mGlu1 pEC50
[% Glu Max SEM]
( SEM)
O
21a
21b
21c
21d
21e
21f
21g
21h
21i
4-Cl
5-Cl
7-Cl
4-Me
4-F
4-Br
4-CF3
4-CN
0.211 [88 4]
>10 [58 1]
0.484 [108 4]
1.07 [89 3]
0.878 [82 4]
0.391 [99 4]
1.13 [105 5]
2.09 [107 9]
>10 [—]
6.75 0.07
>5
N
20a
>10 [—]
>5
>5
6.31 0.1
5.96 0.09
6.05 0.11
6.41 0.10
5.94 0.09
5.67 0.17
>5
O
O
N
20b
>10 [—]
O
O
4-OMe
4-Aza
21j
>10 [—]
>5
21k
21l
21m
7-Aza
4,7-Aza
3,3-diMe
>10 [—]
>10 [—]
6.93 [76 3]
>5
>5
20c
20d
0.93 [108 7]
>10 [-]
6.03 0.14
>5
HN
CO2H
O
5.15 0.09
a
Calcium mobilization mGlu1 assays, values are average of three (n = 3) inde-
pendent experiments performed in triplicate.
HN
O
20e
20f
3.72 [91 9]
5.43 0.19
5.75 0.43
N
Analogs 22 surveyed SAR for three regions: the central phenyl
core, alternative heterocyclic amides and substitutions on the
isoindolinone phenyl ring (Table 3). The removal of the hydrogen
in the central ring (22a–c) led to a similar SAR tendency as the
2-Cl analogs, with 22b having a comparable potency to 21a, sug-
gesting that an asymmetric substitution pattern in the central ring
is not necessary. Other substitutions, such as 2,3-dichloro (22d–f)
and 3-fluoro (22g–i) caused a dramatic loss in potency. This trend
shows how variable and steep the SAR can be in this scaffold, as the
3-fluoro substitution has been an excellent modification to achieve
potent and selective mGlu1 PAM activity in our phthalimide scaf-
fold. As we assessed the replacement of the furan with different
heterocycles in the isoindolinone scaffold, a substantial loss in
potency was found when using 3-substituted picolinamides, and
only the 4-Cl substituted 22h and 22k (direct comparators to
N
1.76 [95 23]
O
N
20g
>10 [—]
>5
N
20h
20i
20j
5.71 [82 8]
9.55 [78 2]
3.62 [99 9]
5.24 0.14
5.02 0.09
5.44 0.14
N
N
N
O
N
O
21a) maintained mGlu1 PAM potency of around 1 lM; while in
20k
2.61 [93 4]
5.58 0.07
N
O
the thiazole (22p–r) congeners the 4-Cl substituted (22q) was
most potent of the group (EC50 = 950 nM). However, these
attempts to engender improved free fraction in the isoindolinone
series failed, and SAR was steep.
20l
0.781 [94 4]
2.47 [107 5]
6.11 0.12
5.88 0.09
N
In conclusion, the continued optimization of the VU0486321
series of mGlu1 PAMs led us to pursue alternatives for the phthal-
imide moiety, plagued with unpredictable plasma instability
issues. Here we identified isoindolinones as biosiosteres for the
phthalimide group. mGlu1 PAMs such as 21a, that retained mGlu1
PAM potency and mGluR selectivity, while also affording plasma
stability, attractive in vitro and in vivo PK profiles and high CNS
penetration (Kp = 1.36). Together, with VU0487351 (21a) and
VU6004909 (5), the field now has mGlu1 PAMs that can serve as
robust in vivo tool compounds to further dissect the therapeutic
potential of selective mGlu1 activation.
N
20m
a
Calcium mobilization mGlu1 assays, values are average of three (n = 3) inde-
pendent experiments performed in triplicate.
rat plasma, as well as rat brain homogenate binding), which was
true for all analogs 21 (fu < 0.01). Therefore, we elected to pursue
a matrix library strategy to identify new isoindolinone-based
mGlu1 PAMs that maintained the overall profile of 21a, while
improving fraction unbound.

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P. M. Garcia-Barrantes et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1869–1872
Table 3
Medicine (to C.W.L.). P.M.G. would like to acknowledge the VISP
program for its support. This work was funded by the William K.
Warren, Jr. Chair in Medicine and the NIH (U54MH084659).
Structures and activities for analogs 22
R1
O
O
7
Het
R2
N
NH
References and notes
4
22
1. Frank, R. A. W.; McRae, A. F.; Pocklington, A. J.; van de Lagemaat, L. N.; Navarro,
P.; Croning, M. D. R.; Komiyama, N. H.; Bradley, S. J.; Challiss, R. A. J.; Armstrong,
J. D.; Finn, R. D.; Malloy, M. P.; MacLean, A. W.; Harris, S. E.; Starr, J. M.; Bhaskar,
S. S.; Howard, E. K.; Hunt, S. E.; Coffey, A. J.; Raganath, V.; Deloukas, P.; Rogers,
J.; Muir, W. J.; Deary, I. J.; Blackwood, D. H.; Visscher, P. M.; Grant, S. G. N. PLoS
One 2011, 6 e19011.
2. Ayoub, M. A.; Angelicheva, D.; Vile, D.; Chandler, D.; Morar, B.; Cavanaugh, J. A.;
Visscher, P. M.; Jablensky, A.; Pfleger, K. D. G.; Kalaydijeva, L. PLoS One 2012, 7
e32849.
3. Cho, H. P.; Garcia-Barrantes, P. M.; Brogan, J. T.; Hopkins, C. R.; Niswender, C.
M.; Rodriguez, A. L.; Venable, D.; Morrison, R. D.; Bubser, M.; Daniels, J. S.;
Jones, C. K.; Conn, P. J.; Lindsley, C. W. ACS Chem. Biol. 2014, 9, 2334.
4. Garcia-Barrantes, P. M.; Cho, H. P.; Niswender, C. M.; Byers, F. W.; Locuson, C.
W.; Blobaum, A. L.; Xiang, Z.; Rook, J. M.; Conn, P. J.; Lindsley, C. W. J. Med.
Chem. 2015, 58. 7959–7571.
5. Garcia-Barrantes, P. M.; Cho, H. P.; Niswender, C. M.; Blobaum, A. L.; Conn, P. J.;
Lindsley, C. W. Bioorg. Med. Chem. Lett. 2015, 25, 5107.
6. Garcia-Barrantes, P. M.; Cho, H. P.; Metts, A. M.; Blobaum, A. L.; Niswender, C.
M.; Conn, P. J.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2016, 26, 751.
7. Vieira, E.; Huwyler, J.; Jolidon, S.; Knoflach, F.; Mutel, V.; Wichmann, J. Bioorg.
Med. Chem. Lett. 2009, 19, 1666.
8. Jones, C. K.; Engers, D. W.; Thompson, A. D.; Field, J. R.; Blobaum, A. L.; Lindsley,
S. R.; Zhou, Y.; Gogliotti, R. D.; Jadhav, S.; Zamorano, R.; Daniels, J. S.; Morrison,
R.; Weaver, C. D.; Conn, P. J.; Lindsley, C. W.; Niswender, C. M.; Hopkins, C. R. J.
Med. Chem. 2011, 54, 7639.
9. Representative experimental (21a, VU0487351). N-(3-Chloro-4-(4-chloro-1-
oxoisoindolin-2-yl)phenyl)-3-methylfuran-2-carboxamide (21a): In a microwave
vial, N-(4-amino-3-chlorophenyl)-3-methylfuran-2-carboxamide (preparation
described in Ref. 4) (100 mg, 0.399 mmol) and K2CO3 (110 mg, 0.798 mmol)
were weighted an dissolved in DMF (2 mL). The reaction was stirred for 10 min
at room temperature followed by addition of 2-(bromomethyl)-3-chloroben-
zoate (126 mg, 0.479 mmol). The reaction was heated in a microwave reactor
for 15 min at 150 °C. The reaction was worked up by addition of water and
successive extractions with DCM. The organic phases were combined and
filtered through a phase separator. Volatiles were evaporated in vacuo and the
crude was purified by trituration with cold MeOH. A cream solid was obtained
(84.8 mg, 53%). 1H NMR (400.1 MHz, CDCl3) d (ppm): 8.16 (1H, s), 8.02 (1H, d,
J = 2.3 Hz), 7.87 (1H, J = 7.4 Hz), 7.59 (2H, m), 7.51 (1H, t, J = 7.7 Hz), 7.38 (2H,
m), 6.42 (1H), 4.78 (2H, s), 2.47 (3H, s). 13C NMR (100.6 MHz, CDCl3) d (ppm):
167.3, 157.2, 142.8, 141.4, 139.8, 138.6, 133.9, 133.1, 131.9, 130.7, 130.1, 129.9,
129.8, 129.2, 122.8, 121.1, 118.7, 116.0, 51.4, 11.2.
Cpd
R1
R2
Het
hmGlu1 EC50
[% Glu Max SEM]
(
l
M)a
mGlu1 pEC50
( SEM)
O
O
O
O
22a
22b
22c
22d
22e
H
H
1.56 [94 12]
5.81 0.31
6.61 0.11
6.45 0.11
>5
H
4-Cl
7-Cl
H
0.245 [99 3]
0.353 [101 3]
>10 [—]
H
2,3-diCl
2,3-diCl
O
O
O
4-Cl
>10 [—]
>5
22f
22g
22h
22i
2,3-diCl
3-F
7-Cl
H
6.52 [81 6]
>10 [—]
5.18 0.09
>5
O
3-F
4-Cl
7-Cl
1.26 [86 6]
>10 [—]
5.90 0.15
>5
O
3-F
N
N
22j
2-Cl
2-Cl
H
>10 [—]
>5
22k
4-Cl
1.36 [97 6]
5.84 0.14
10. In vitro molecular pharmacology. Tetracycline-tested fetal bovine serum (FBS)
was purchased from Atlanta Biologicals (Lawrenceville, GA), and all other
tissue culture reagents and Fluo-4-acetoxymethylester (Fluo-4-AM) were
purchased from Life Technologies (Carlsbad, CA). Tetracycline hydrochloride
N
22l
2-Cl
2-Cl
7-Cl
H
>10 [78 2]
>10 [—]
>5
>5
(Sigma), L-glutamic acid (Tocris, Minneapolis, MN), and (S)-3,5-dihydroxy
N
phenylglycine (DHPG) (Abcam, Cambridge, MA). EZ-Link Sulfo-NHS-SS-Biotin
and NeutrAvidin agarose beads (Pierce Biotechnology, Rockford, IL).
Tetracycline-inducible human mGlu1 WT-T-RExTM-293 cells (Wu et al.,
Science 2014) were cultured at 37 °C in Dulbecco’s Modified Eagle Medium
22m
Cl
N
(DMEM) growth medium containing 10% Tet-tested FBS, 2 mM
20 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate,
antibiotic/antimycotic, 100 g/mL hygromycin and 5 g/mL blasticidin in the
L-glutamine,
22n
22o
22p
22q
2-Cl
2-Cl
2-Cl
2-Cl
4-Cl
7-Cl
H
1.27 [93 5]
>10 [51 3]
>10 [33 6]
0.954 [66 4]
5.89 0.12
>5
Cl
Cl
l
l
N
presence of 5% CO2. To determine the potency of mGlu1 PAMs in calcium
assays, Ca flux was measured as previously described in ACS Chem. Biol. 2014, 9,
2334. Briefly, the day before the assay, human mGlu1 WT-T-RExTM-293 cells
were plated in black-walled, clear-bottomed, poly-
D-lysine coated 96-well
N
plates at 80,000 cells/100 L assay medium (DMEM supplemented with 10%
l
>5
dialyzed FBS, 20 mM HEPES, and 1 mM sodium pyruvate) containing 50 ng/mL
of tetracycline to induce mGlu1 expression. The next day, media was removed
and the cells were incubated with 50 lL of 1.15 lM Fluo-4 AM dye solution
prepared in assay buffer (buffer (Hank’s balanced salt solution, 20 mM HEPES,
and 2.5 mM probenecid) for 45 min at 37 °C, the dye was removed and
S
N
4-Cl
6.02 0.12
S
replaced with 45 lL of assay buffer. Then, calcium flux was measured using
N
Flexstation II (Molecular Devices, Sunnyvale, CA). Compounds serially diluted
at half log concentrations in DMSO were further diluted in assay buffer. The
compounds or DMSO vehicle were added to cells and incubated for 2.5 min and
an EC20 concentration of glutamate was added and incubated for 1 min. An
ECmax concentration of glutamate was also added to cells that were incubated
with DMSO vehicle to accurately calculate the EC20 calcium response. Data
were normalized by subtracting the basal florescent peak before EC20 agonist
addition from the maximal peak elicited by EC20 agonist and PAMs. Using
GraphPad Prism 5.0, the concentration response curves were generated and the
potencies of the mGlu1 PAMs were determined.
22r
2-Cl
7-Cl
4.69 [69 2]
5.32 0.73
S
a
Calcium mobilization mGlu1 assays, values are average of three (n = 3) inde-
pendent experiments performed in triplicate.
Acknowledgments
11. Gentry, P. R.; Kokubo, M.; Bridges, T. M.; Byun, N.; Cho, H. P.; Smith, E.; Hodder,
P. S.; Niswender, C. M.; Daniels, J. S.; Conn, P. J.; Lindsley, C. W.; Wood, M. R. J.
Med. Chem. 2014, 57, 7804.
We thank William K. Warren, Jr. and the William K. Warren
Foundation who funded the William K. Warren, Jr. Chair in