Discovery, synthesis, and structure-activity studies of tetrazole based growth hormone secretagogues

Article abstract of DOI:10.1016/j.bmcl.2007.07.099

A novel class of Growth Hormone Secretagogues (GHS), based on a tetrazole template, has been discovered. In vitro SAR and in vivo potency within this new class of GHS are described. The tetrazole 9q exhibits good oral bioavailability in rats and dogs as w

Full text of DOI:10.1016/j.bmcl.2007.07.099

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5928–5933
Discovery, synthesis, and structure–activity studies of tetrazole
based growth hormone secretagogues
a,
a
a
a
*
´
´
Andres S. Hernandez, Peter T. W. Cheng, Christa M. Musial, Stephen G. Swartz,
Rocco J. George,b Gary Grover,b Dorothy Slusarchyk,b R. Krishna Seethala,b
Mark Smith,b Kenneth Dickinson,b Leah Giupponi,b Daniel A. Longhi,b Neil Flynn,b
Brian J. Murphy,b David A. Gordon,b Scott A. Biller,a
Jeffrey A. Robla and Joseph A. Tinoa,*
aDiscovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
bMetabolic Disease Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
Received 4 June 2007; revised 24 July 2007; accepted 24 July 2007
Available online 23 August 2007
Abstract—A novel class of Growth Hormone Secretagogues (GHS), based on a tetrazole template, has been discovered. In vitro
SAR and in vivo potency within this new class of GHS are described. The tetrazole 9q exhibits good oral bioavailability in rats
and dogs as well as efficacy following an oral 10 mg/kg dose in dogs. Solution and solid phase protocols for the synthesis of tetrazole
based GHS have been developed.
Ó 2007 Elsevier Ltd. All rights reserved.
Growth Hormone Secretagogues (GHS) induce growth
hormone (GH) release upon binding to the GHS recep-
tor, a G-protein-coupled receptor located primarily in
the pituitary and the hypothalamus. The first GHS re-
ported were the growth hormone releasing peptides
(GHRPs), such as the hexapeptide GHRP-6.1 Subse-
quently, small, peptidomimetic, orally bioavailable
GHS, such as MK-677, were reported2,3 prior to identi-
fication of the natural GHS-receptor ligand, ghrelin in
1999.4,5
NH2
NH2
NH
O
O
NH
Tetrazole
Isostere
O
O
O
N
N
N
N
N
O
N S
Linker
O
H-Bond
Acceptor
MK-677
Figure 1. MK-677 and proposed tetrazole based chemotype.
MK-6776 (Fig. 1) contains a dipeptide formed by O-ben-
zyl-D-serine and 2-methyl alanine. The carboxy-termi-
nus of this dipeptide embedded within MK-677 is
connected to a H-bond acceptor through a lipophilic lin-
ker (Fig. 1). Published ghrelin structure–activity studies
suggest that this dipeptide corresponds to the N-termi-
nus of the GHRPs and ghrelin.7 This paper describes
our design of a novel series of tetrazole based GHS ago-
nists. We maintained the dipeptide fragment present in
MK-677 but replaced the amide component with a tetra-
zole isostere. Disubstituted tetrazoles, which can exist as
1H or 2H regioisomers, are known bioisosteres for car-
boxylic acids and their derivatives.8 In addition to struc-
tural novelty, the two possible isomeric cores generated
upon replacement of the carboxy group by a tetrazole
ring could exhibit different ADME properties from that
of MK-677. Although modeling suggested that
structural superposition of the 2H-tetrazole core with
MK-677 was preferred, both the 1H- and 2H-tetrazole
analogs were synthesized and tested in in vitro assays.
Our synthesis leading to 1H- and 2H-tetrazole analogs
is depicted in Scheme 1. Coupling of N-Boc-O-benzyl-
D-serine, via its mixed carbonic anhydride,9 and
Keywords: Growth hormone secretagogues; Tetrazole.
*
Corresponding authors. Tel.: +1 609 818 7127; fax: +1 609 818 6810
(A.S.H.); e-mail: andres.hernandez@bms.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.099

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A. S. Hernandez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5928–5933
5929
NHBOC
NHBOC
1. NMM, iBuO2CCl
TMSN3
NHBOC
N
BnO
BnO
BnO
CO2H
CN
CN
2.
O
N
H
Ph3P, DEAD
(3 eq. ea., 48h)
75%
N
N
H2N
97%
CN
N
1
2
1. HCl
81%
2. BOC-Aib, EDAC, HOAT
NHR2
NHBOC
NHR2
O
O
O
1. RBr (5), Cs2CO3 or
ROH (6), Ph3P, DEAD
NH
NH
N R1
NH
N R
BnO
BnO
BnO
H+
+
2. Isomer Separation
(SiO2 column)
N
N
N
N
N
N
N
N
N
N
R
3, R1= -(CH)2-CN
4, R1= H
8, R2= BOC
10, R2= H
7, R2= BOC
9, R2= H
NaOH
(95%)
H+
Scheme 1. Synthesis of 1H- and 2H-tetrazoles.
3-aminopropionitrile furnished the amide 1 in 97%
yield. Amide 1 reacted with triphenylphosphine, diethyl
azodicarboxylate (DEAD), and azidotrimethylsilane9,10
to form tetrazole 2 in 75% yield. Boc deprotection fol-
lowed by amino-acid coupling with N-Boc-2-methyl
alanine generated amide 3 in 81% yield for the two
steps. Removal of the cyanoethyl protecting group
yielded the 5-substituted tetrazole 4 in 95% yield.
Alkylation of tetrazole 4 with alkyl bromides 5 or via
Mitsunobu conditions with alcohols 6 produced a mix-
ture of isomeric 1H-/2H-tetrazoles 7/8 (in >60% yield)
ranging in composition from 1:1 to predominantly
the 2H-tetrazole.11 Cleavage of the isomeric Boc carba-
mates 7 or 8 yielded the corresponding amines 9 or 10,
in high yield. Although the linker moiety and/or the
H-bond acceptor were occasionally commercially avail-
able as alkyl bromides 5, in most instances this compo-
nent came from alcohols 6 prepared as depicted in
Scheme 2.
We postulated that an alkyl tethered phenyl as the linker
joining the tetrazole ring to the methanesulfonamide
(MeSO2N) moiety maximized the probability of emulat-
ing the putative favorable H-bond acceptor receptor
interactions of MK-677 since three different positional
isomers are possible for the resulting aryl sulfonamides.
Variation of the site of attachment as well as the length
and conformational flexibility of the alkyl tether
generated tetrazoles 9 and 10. In vitro characterization
revealed that these ligands typically exhibited 5- to 20-
fold greater functional activity12 than binding affinity13
for the GHS-receptor (Table 1). This fact and the find-
ing that all these tetrazoles are full agonists prompted
us to analyze the following SAR based on functional
activity.
For the 1H series 9a–9f, analogs containing a 1- or 2-car-
bon linker between the tetrazole and the distal phenyl
ring were generally equipotent even if the phenyl ring
was incorporated within a polyaromatic system (e.g. indo-
line 9f; Table 1). Furthermore, the presence of the Me-
SO2NH moiety appeared to confer no advantage
(comparison of 9a–9b or 9c) and could induce a fourfold
decrease in potency (9d). However, further elongation to
a 3-carbon linker generated the phenyl–propyl 9g and the
more constrained phenylpropenyl 9h analogs, exhibiting,
respectively, 5- and 10-fold improved EC50 values rela-
tive to 9a. Functional activity of 9i relative to 9g was
not enhanced upon inclusion of an ortho MeSO2NMe
substituent. However, the conformational constraint im-
posed by linking the two appendages of 9i to generate the
indolyl counterpart 9j increased potency by fivefold.
HO
HO
1. TMSCl, ImH
2. MsCl. Et3N
6b: (1,4), n=1
6c: (1,3), n=1
6d: (1,2), n=1
6e: (1,2), n=2
( )n
( )n
O
S
NBOC
O
3. BOC2O, DMAPa
4. SiO2, MeOH
NH2
CO2Me
OH
1. NaH, MsCl
2. Ca(BH4)2
6f
N
N
S O
H
O
1. NaH, MsCl
2. Ca(BH4)2
OH
6i
6j
N
H
O
N
S O
3. MeI, Cs2CO3
In a finding counter to the original molecular modeling
prediction, the isomeric 2H analogs 10a–f and 10h–j
(Table 1) were up to 40-fold less potent agonists than
the corresponding 1H isomers. Unlike the 1H series,
tether elongation to a three carbon spacer (10h) did
not significantly improve potency relative to the parent
10a. Just as was observed for the 1H series, the presence
of both the three carbon spacer and the MeSO2NMe
substituent for the 2H series enhanced potency the most;
however, the SAR response was only about 10-fold (10i
or 10j versus 10a) as compared to 25-fold for the 1H
series.
O
OH
OH
1. TMSCl, ImH
2. NaH, MsCl
N
H
3. H+ work-up
N
S O
O
Scheme 2. Synthesis of alcohols 6. aBoc protection prevents interfer-
ence of the sulfonamide function in the subsequent Mitsunobu
reaction. This Boc group is removed along with the Boc group
protecting the amine.

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A. S. Hernandez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5928–5933
5930
Table 1. In vitro activity of tetrazole analogs 9 and 10
Table 1 (continued)
b
EC50 (nM)
Compound
R
Subst.
Kia (nM)
NH2
NH2
O
O
9o
10o
1-H
2-H
3120
>5000
35
1860
CN
NH
N R
NH
BnO
BnO
9p
10p
1-H
2-H
4170
>5000
29
1520
N
N
N
N
N
CN
CN
N
N
R
9
10
9q
1-H
2940
30
a See Ref. 13 for detailed description.
b See Ref. 12 for detailed description.
b
Compound
R
Subst. Kia (nM) EC50 (nM)
9a
10a
1-H
2-H
1900
4750
99
1120
To assess the functional activity of an analog containing
an H-bond acceptor that, unlike MK-677, has no direct
attachment to the phenyl ring, the benzylic carbon of 9a
was substituted with a CO2Me moiety to generate 9k.
The fivefold improvement in potency of 9k over 9a or
any of the benzyl sulfonamides 9b–9d indicates that di-
rect attachment of the H-bond acceptor to the phenyl
ring is not essential for potency. Moreover, analogs
without the phenyl ring (e.g. the pentyl sulfonamide 9l
and tetrazole 9m) exhibited activities similar to those
of benzyl analog 9a–9d. This data demonstrated that
either an appropriately tethered phenyl ring (i.e. 9g) or
an H-bond acceptor (9l) connected to the tetrazole scaf-
fold afforded significant GHS-receptor activity. The five-
fold decrease in potency of 9l relative to that of 9i is
most likely due to the loss of conformational constraint
imposed by the phenyl ring. Increased lipophilicity en-
hanced in vitro GHS-receptor activity as exemplified
by eightfold greater potency of the benzyl carbamate
9n relative to 9l.
9b
10b
1-H
2-H
>5000
>5000
259
986
O O
S
N
H
H
N
9c
10c
1-H
2-H
>5000
>5000
116
868
S
O
O
S
O
9d
10d
1-H
2-H
4450
>5000
390
426
HN
O
S O
O
HN
9e
10e
1-H
2-H
2200
2660
67
4110
9f
10f
1-H
2-H
1590
>5000
147
546
N
S
O
O
The emerging SAR pattern favoring disubstituted 1H-
tetrazoles prompted the development of a regiospecific
synthetic route summarized in Scheme 3. Starting with
the appropriate primary amine 11, mixed anhydride
coupling with N-Boc-O-benzyl-D-serine followed by tet-
razole formation, Boc deprotection, and aminoacid cou-
pling with N-Boc-2-methyl alanine afforded the
disubstituted 1H-tetrazole 12. Cleavage of the Boc car-
bamate 12 provided target amine 9, as its TFA or HCl
salt. The ethyl nitrile 9q, the phenylpropyl analog 9g,
and the n-pentyl Cbz carbamate 9n were obtained by
this last procedure. The methanesulfonamide 12l was
prepared from Cbz carbamate 12n by hydrogenolysis
followed by sulfonylation. Addition of trimethyltin
azide10 to nitrile 12q generated the bis-tetrazole 12m.
9g
1-H
200
17
9h
10h
1-H
2-H
190
>5000
12
517
9i
10i
1-H
2-H
1720
2390
14
107
N
S
O
O
9j
10j
1-H
2-H
970
1270
4
150
N
S
O
O
To ascertain the optimal length of the tether between the
tetrazole and an H-bond acceptor, nitriles 9o–9q were
evaluated. However, since both the binding affinities
9k
10k
1-H
2-H
470
>5000
20
3170
MeO2C
O O
S
9l
1-H
1-H
>5000
5000
75
NHR1
N
H
O
1. NMM, iBuO2CCl, RNH2 (11)
2. TMSN3, Ph3P, DEAD
N
N
9m
347
NHBOC
CO2H
NH
HN
N
BnO
BnO
3. TFA or HCl
4. BOC-Aib, EDAC, HOAT
N
N
N R
N
NH
9n
1-H
320
10
12, R1= BOC
H+
O
O
9, R1= H
Scheme 3. Synthesis of 1H-tetrazoles.

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A. S. Hernandez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5928–5933
5931
Table 2. In vitro activities of 1H-tetrazole-esters and -amides 19a–19i
and functional activity are essentially the same for all
three compounds, it is apparent that spacing for the ni-
trile is not relevant unlike the case of the aryl-bearing
analogs.
NH2
O
R1 NH
N
N
()n
O
N
The encouraging in vitro activities exhibited by nitriles
9o–9q prompted further SAR exploration of 1H-tetra-
zoles formed from other aminoacids and/or linked to
different H-bond acceptor groups by simple alkyl chains.
In order to enable the different permutations between
aminoacid residues (R1), alkyl chains (R2), and H-bond
donor function (COR3, R3= OMe or NHR), a solid
phase synthesis (Scheme 4) of disubstituted 1H- tetraz-
oles was devised.14 Anchoring of the D-aminoacid bear-
ing a selected residue (R1) to a previously activated resin
bound (tert-alkoxycarbonyl)imidazole 1415 generated
the corresponding resin bound aminoacid 15. The alkyl
linker (R2) was introduced by condensation of 15 with
an aminoalkyl methylester to yield immobilized 16. Tet-
razole formation,16 followed by aminolysis (RNH2) of
the methyl ester 16, afforded the resin bound tetrazole-
amide 17 (R3 = NHR). Acidic cleavage of the resin
released amine 18. Aminoacid coupling of 2-methyl-
alalanine with 18 followed by Boc deprotection yielded
the target amines 19 as their HCl salts.
R3
N
Compound R1
N
2
R3
Kia
EC50
b
(nM)
43
O
O
O
O
O
O
O
19a
–OMe
3380
19b
19c
19d
19e
19f
19g
19h
19i
3
2
3
2
2
2
2
2
–OMe
2380
2190
1780
1070
55
42
30
51
27
59
49
–NHnBu
–NHnBu
–NHCH2Ph
–NH(CH2)2Ph 780
–NH(CH2)3Ph 820
The profile of all the O-benzyl-D-serine derived tetra-
zoles tabulated in Table 2 shows that ester and amide
functionality are equivalent replacements for the nitrile
as H-bond acceptors. This series differs from that previ-
ously discussed as there appears to be little dependency
on lipophilicity and spacer length. Comparison of the
phenylalkyl amides 19e–19g to the butyl amide 19c re-
veals no change in potency as a consequence of increas-
ing lipophilicity. The equivalence in functional and
binding activities of esters 19a and 19b or the amides
19c and 19d reveals the insensitivity to two versus three
–NHnBu
–OMe
1370
>5000 200
N
H
a See Ref.13 for detailed description.
b See Ref.12 for detailed description.
1. MeOTf
O
O
O
O
2. Et3N
O
O
N
N
R1 NH
1
3.
R
NH2
carbon tethers. The equivalent potencies of the O-benzyl
serine derivative 19c and the corresponding homophe-
nylalanine 19h suggest that the ether moiety does not
participate in H-bonding to the receptor. Finally, in this
series, the tryptophan-derived 19i is fourfold less potent
than its O-benzyl counterpart 19a. Overall, these tetra-
zole-esters and -amides have similar in vitro activities
to that of tetrazole nitrile 9q.
CO2H
CO2H
14
15
R2
H2N CO2Me
EDAC, HOAT
1. TMSN3,
Ph3P, DEAD
O
O
O
O
O
O
R1 NH
R1 NH
2. RNH2,
R2
N
N
n-BuN4CN
N R2
O
N
H
CO2Me
The compounds in Table 1 can be divided into three dis-
tinct classes of 1H-tetrazole agonists: (a) compounds
featuring an H-bond acceptor linked to the tetrazole
by a lipophilic tether such as 9j, (b) compounds linked
to only a lipophilic group such as 9h, and (c) compounds
with a H-bond acceptor attached to the tetrazole by an
alkyl chain such as 9q. Results from in vivo evaluation
of three representative compounds, 9j, 9h, and 9q, in
an acute anesthetized IV rat model17 are summarized
in Table 3.
R3
N
16
O
17
3
M
HCl/
NH2
CH2Cl2, MeOH
O
R1 NH
R1 NH2
1. BOC-Aib,
EDAC, HOAT
N
N
N
N R2
N R2
N
N
R3
2. 3 HCl/
M
R3
N
CH2Cl2, MeOH
O
O
19
18
In spite of lower in vitro functional activity, the propio-
nitrile 9q exhibited superior in vivo activity to that of the
Scheme 4. Solid supported synthesis of 1H-tetrazoles 19.

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A. S. Hernandez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5928–5933
5932
Table 3. In vitro and in vivo activity in the acute anesthetized IV rat
model of 1H-tetrazoles
2. DeVita, R. J.; Schoen, W. R.; Fischer, M. H.; Fontier, A.
J.; Pisano, J. M.; Wyvratt, M. J.; Cheng, K.; Chan, W.-S.;
Butler, B. S.; Hickey, G. J.; Jacks, T. M.; Smith, R. G.
Bioorg. Med. Chem. Lett. 1994, 4, 2229.
Compounds Structure
EC50 ED50
(nM)a (lmol/kg)b
CLogP
3. Patchett, A. A.; Nargund, R. P.; Tata, J. R.; Chen, M.-H.;
Barakat, K. J.; Johnston, D. B. R.; Cheng, K.; Chan, W.
W.-S.; Butler, B.; Hickey, G.; Jacks, T.; Schleim, K.; Pong,
S.-S.; Chaung, L.-Y. P.; Chen, H. Y.; Frazier, E.; Leung,
K. H.; Chiu, S.-H. L.; Smith, R. G. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 7001.
4. Ghrelin is a 28-amino acid peptide that displays a unique
octanoyl-serine residue and is secreted in the stomach.
5. Kojima, M.; Hosoda, H.; Date, Y. Nature 1999, 402,
656.
NH2
O
NH
BnO
9j
4
>17.4
1.8
O O
S
N
N
N
N
N
NH2
O
6. (a) Svensson, J. Exp. Opin. Ther. Patents 2000, 10, 1071;
(b) Carpino, P. A. Exp. Opin. Ther. Patents 2002, 12, 1599.
7. Dodge, J. A.; Heiman, M. L. Annu. Rep. Med. Chem.
2003, 38, 81.
NH
N
BnO
9h
12
26.4
2.5
N
N
N
8. (a) Thornber, C. W. Chem. Soc. Revs. 1979, 8, 563; (b)
Singh, H.; Chawla, A. S.; Kapoor, V. K.; Paul, D.;
Malhotra, R. K. Prog. Med. Chem. 1980, 17, 151.
9. De Lombaert, S.; Blanchard, L.; Tan, J.; Sakane, Y.;
Berry, C. Bioorg. Med. Chem. Lett. 1995, 5, 145.
10. Duncia, J. V.; Pierce, M. E.; Santella, J. B., III J. Org.
Chem. 1991, 56, 2395.
NH2
O
NH
N
9q
30
8.5
À0.3
BnO
N
N
11. Isomer separation was usually achieved at this step by
column chromatography on silica gel. For a few analogs,
1H/2H isomeric mixtures were more easily separated by
CN
N
a See Ref. 12 for detailed description.
b See Ref. 17 for detailed description.
1
preparative HPLC. HNMR easily distinguished between
the 1H and 2H isomers by comparison of the Dd between
the methyls of the 2-aminoisobutyric fragment. The Dd is
significantly larger in the 1H isomer than in the 2H isomer.
12. H4 glioma cells in which expression of the endogenous
human GHS receptor was enhanced by RAGE-activation
were utilized for both the functional and binding assays.
For details about random Activation for GENE EXPRE-
SION (RAGE)TM technology (Athersys, Inc.), see: Harr-
inton, J. J.; Sherf, B.; Rundlett, S.; Jackson, P. D.; Perry,
R.; Cain, S.; Leventhal, C.; Thorton, M.; Ramachandran,
R.; Whittington, J.; Lerner, L.; Costanzo, D.; McElligott,
K.; Boozer, S.; Mays, R.; Smith, E.; Veloso, N.; Klika, A.;
Hess, J.; Cothren, K.; Lo, K.; Offenbacher, J.; Danzig, J.;
Ducar, M. Nat. Biotechnol. 2001, 19, 440, The EC50 was
measured by determining intracellular calcium concentra-
tion in a FLIPR assay with Ghrelin as having 100%
intrinsic functional activity. All EC50 values are averages
of at least three measurements.
13. Binding affinities were determined by 12-point dose–
response curve analysis using membranes from RAGE-
based GHSR1a expressing cells. Typically, 0.5 ug mem-
brane was incubated in the presence of 0.02 nM [125I]
Ghrelin in 25 mM Hepes (pH 7.2) with 10 mM MgCl2,
2 mM EGTA, and 0.1% BSA in a total volume of 0.2 ml
for 1 h in the presence of increasing concentrations of test
compound. Reactions were terminated by rapid vacuum
filtration over GFB filterplates coated with 0.1% poly-
ethylenimine, and plates were then washed with 0.8 ml of
binding buffer and radioactivity subsequently measured by
scintillation spectroscopy on a TopCount (Perkin-Elmer)
scintillation counter. Curves were fit by nonlinear regres-
sion analysis using a four parameter logistic equation, and
Ki values were determined using the Cheng-Prusoff equa-
tion. Compounds were routinely tested on three separate
occasions.
other two more liphophilic classes of tetrazoles. This re-
sult may be attributed to a potentially better ADME
profile of the polar smaller agonist 9q. The oral bioavail-
ability of 9q was 56% and 76% in rats and dogs, respec-
tively. When administered orally to beagle dogs at a
10 mg/kg dose, 9q increased GH mean peak levels to
30 ng/mL compared to 0 ng/mL for vehicle control.
Although maximum GH levels occurred between 40
and 45 min post-dose, elevated plasma concentrations
of GH persisted for up to 120 min after dosing.
In summary, several novel series of GHS-receptor ago-
nists based on a novel tetrazole template have been de-
scribed. Reliable solution and solid phase protocols
have been developed for the synthesis of the three re-
lated sets of analogs 9, 10, and 19. The SAR of the linker
and H-bond acceptor were explored. The lead analog 9q
exhibited good oral bioavailability in rats and dogs;
moreover, it was orally efficacious in dogs at the
10 mg/kg dose. Further efforts to improve the in vitro
and in vivo potency of this 1H-tetrazole chemotype will
be reported elsewhere.
Acknowledgments
We thank Dr. William Washburn, Dr. Scott Priestley,
and Dr. Robert Zahler for their suggestions on the
manuscript.
14. Full experimental details will be disclosed in a manuscript
which is in preparation.
References and notes
´
15. (a) Hernandez, A. S.; Hodges, J. C. J. Org. Chem. 1997,
62, 3153; (b) Wilson, M. W.; Hernandez, A. S.; Calvet, A.
´
1. Bowers, C. Y.; Momany, F. A.; Reynolds, G. A.; Hong,
A. Endocrinology 1984, 114, 1537.
P.; Hodges, J. C. Mol. Diversity 1998, 3, 95.

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A. S. Hernandez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5928–5933
5933
16. (a) The removal of the large amounts of triphenylphos-
phine oxide and diethyl hydrazide generated as by-
products during the tetrazole formation step is greatly
simplified by this solid phase methodology. (b) A portion
of the resin bound tetrazole ester was cleaved and coupled
to Boc-Aib followed by Boc cleavage in order to evaluate
the in vitro activity of the tetrazole ester (19, R3 = OMe).
17. Acute anesthetized rat model: fasted male Wistar rats
(200–250 g) were anesthetized via intraperitoneal injection
with ketamine (30 mg) and xylazine (10 mg) per kg body
weight. Vehicle (10% EtOH/water with 0.9% saline V/V)
or the GH secretagogue was administered IV at a volume
of 1 mL/kg. After 15 min (the Tmax for GH in this model),
1.5 mL blood samples were drawn from the abdominal
aorta. Plasma samples were then analyzed for rat GH by
radioimmunoassay using a modification of the kit supplied
by the National Pituitary hormone Center (Dr. A. Parlow,
Harbor-UCLA Medical Center, Los Angeles, CA).