Wet granulation formulation of a growth hormone secretagogue

The present invention relates to a pharmaceutical composition and a process for the preparation of a tablet containing a growth hormone secretagogue as the active ingredient. The tablet is prepared by forming a powder blend of the active ingredient N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-1'- yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methyl-propanamide, or a pharmaceutically acceptable salt thereof, in particular the methanesulfonate salt, with a binder/diluent, a first diluent, a second diluent, a first portion of a disintegrant, and a lubricant; wet granulating the powder blend with a solution of ethanol/water to form granules; drying the granules to remove the ethanol/water; adding a second portion of a disintegrant; lubricating the granules; and compressing the dried granules into the desired tablet form. The present invention further relates to a novel amorphous form of the compound N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-1'- yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which is produced directly as a result of the process of tablet formulation.

FIELD OF THE INVENTION 
The present invention relates to solid dosage formulations comprising a 
growth hormone secretagogue and process for their manufacture. More 
specifically, the invention relates to a wet granulation formulation of a 
growth hormone secretagogue compound. The present invention further 
relates to an amorphous form of a growth hormone secretagogue, processes 
for its preparation and uses thereof. 
BACKGROUND OF THE INVENTION 
Growth hormone, which is secreted from the pituitary, stimulates growth of 
all tissues of the body that are capable of growing. In addition, growth 
hormone is known to have the following basic effects on the metabolic 
processes of the body: (1) Increased rate of protein synthesis in all 
cells of the body; (2) Decreased rate of carbohydrate utilization in cells 
of the body; (3) Increased mobilization of free fatty acids and use of 
fatty acids for energy. A deficiency in growth hormone secretion can 
result in various medical disorders, such as dwarfism. 
Various ways are known to release growth hormone. For example, chemicals 
such as arginine, L-3,4dihydroxyphenylalanine (L-DOPA), glucagon, 
vasopressin, and insulin induced hypoglycemia, as well as activities such 
as sleep and exercise, indirectly cause growth hormone to be released from 
the pituitary by acting in some fashion on the hypothalamus perhaps either 
to decrease somatostatin secretion or to increase the secretion of the 
known secretagogue growth hormone releasing factor (GRF) or an unknown 
endogenous growth hormone-releasing hormone or all of these. 
In cases where increased levels of growth hormone were desired, the problem 
was generally solved by providing exogenous growth hormone or by 
administering GRF or a peptidal compound which stimulated growth hormone 
production and/or release. In either case the peptidyl nature of the 
compound necessitated that it be administered by injection. Initially the 
source of growth hormone was the extraction of the pituitary glands of 
cadavers. This resulted in a very expensive product and carried with it 
the risk that a disease associated with the source of the pituitary gland 
could be transmitted to the recipient of the growth hormone. Recombinant 
growth hormone has become available which, while no longer carrying any 
risk of disease transmission, is still a very expensive product which must 
be given by injection or by a nasal spray. 
Other compounds have been developed which stimulate the release of 
endogenous growth hormone such as analogous peptidyl compounds related to 
GRF or the peptides of U.S. Pat. No. 4,411,890. These peptides, while 
considerably smaller than growth hormones are still susceptible to various 
proteases. As with most peptides, their potential for oral bioavailability 
is low. Non peptidal growth hormone secretagogues with a benzolactam 
structure are disclosed e.g., in U.S. Pat. Nos. 5,206,235, 5,283,241, 
5,284,841, 5,310,737, 5,317,017, 5,374,721, 5,430,144, 5,434,261, 
5,438,136 and PCT Publications WO 95/03289, WO 95/03290, WO 95/09633. 
Other growth hormone secretagogues are disclosed in PCT Patent 
Publications WO 94/11012, WO 94/13696, WO 94/19367, WO 95/13069 and WO 
95/14666. 
In particular, Examples 18, 19 and 55 of U.S. Pat. No. 5,536,716 (PCT 
Patent Publication WO 94/13696) and Proc. Natl. Acad. Sci. USA, 92, 
7001-7005 (July 1995) disclose the compound 
N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-1'- 
yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide, and 
salts thereof, especially the methanesulfonate salt, which has the 
structure: 
##STR1## 
This compound is a growth hormone secretagogue which stimulates the release 
of growth hormone in humans and animals. This property can be utilized to 
promote the growth of food animals to render the production of edible meat 
products more efficient, and in humans, to treat physiological or medical 
conditions characterized by a deficiency in growth hormone secretion, and 
to treat medical conditions which are improved by the anabolic effects of 
growth hormone. 
U.S. Pat. No. 5,536,716 and PCT Patent Publication WO 94/13696 disclose 
methods for preparing this compound (see Examples 18, 19 and 55). In 
particular, Example 55 states that the compound prepared by 
recrystallization from ethyl acetate-ethanol-water had a melting point of 
166-168.degree. C. Proc. Natl. Acad. Sci. USA, 92, 7001-7005 (July 1995) 
notes that this compound isolated as a monohydrate had a melting point of 
168-170.degree. C. 
Standard methods for tablet formulation of the active ingredient such as 
direct compression suffer from problems. In particular, this compound is 
relatively unstable in standard pharmaceutical formulations. In addition, 
this compound as a bulk drug suffers from poor flow properties, 
nevertheless, wet granulation was discovered to overcome these 
difficulties preparing tablet formulations. Tablets prepared by the wet 
granulation method produced excellent content uniformity, coupled with 
suitable tablet dissolution and stability. The tablets of the present 
invention, prepared by wet granulation, possessed good hardness at normal 
machine pressures. 
The present invention is also concerned with pharmaceutical formulations 
prepared by the subject process and their use in the treatment of certain 
disorders and diseases. 
Exemplifying the present invention is the process comprising the steps of: 
(1) forming a powder blend of the active ingredient with a binder/diluent, 
a first diluent, a second diluent, and a disintegrant, from 2 to 25 
minutes using a mixer; 
(2) wet granulating the powder blend by adding a solution of ethanol/water 
to the powder blend while mixing over a 1 to 30 minute period to form 
granules; 
(3) drying the granules to remove the ethanol/water with heated air in a 
fluid bed dryer or tray dryer for 10 minutes to 24 hours; 
(4) milling the dried granules to a uniform size; 
(5) adding and blending a disintegrant with the dried milled particles for 
2 to 30 minutes; 
(6) adding and blending a lubricant to the mixture containing the 
disintegrant for 30 seconds to 20 minutes; and 
(7) compressing the lubricated granules mixture into a desired tablet form. 
Further illustrating the invention is the process wherein the active 
ingredient is 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl)-2-amino-2-methylpropanamide 
methanesulfonate. 
Additional illustrations of the invention include the process wherein: the 
binder/diluent is pregelatinized starch; the first diluent is 
microcrystalline cellulose; the second diluent is calcium phosphate 
dibasic; the disintegrant is croscarmellose sodium; and the lubricant is 
magnesium stearate. Preferably, the solution of ethanol/water is in the 
range of 0% to 80% ethanol in water (w/w), more preferably in the range of 
5% to 75% ethanol/water (w/w) and even more preferably approximately 25% 
ethanol/75% water (w/w). 
A further illustration of the invention is the foregoing process further 
comprising the step of applying a coating to the tablet. More particularly 
illustrating the invention is the process wherein coating the tablet is 
accomplished by: 
(1) dry blending titanium dioxide (optionally mixed with talc) with 
hydroxypropyl methylcellulose and hydroxypropyl cellulose to form a dry 
powdered blend; 
(2) adding the dry powdered blend to water to form a slurry; 
(3) adding water to the slurry with stirring to form a suspension; and 
(4) applying the suspension to the tablets. 
More specifically exemplifying the invention is the process comprising the 
steps of: 
(1) forming a powder blend of the active ingredient with pregelatinized 
starch, microcrystalline cellulose, calcium phosphate dibasic, and 
croscarmellose sodium, in a mixer for about 3 to 25 minutes; 
(2) wet granulating the powder blend by adding a solution of 25% 
ethanol/75% water (w/w) to the powder blend while mixing over a 1 to 30 
minute period to form granules; 
(3) drying the granules on a tray dryer or a fluid bed dryer for about 1 to 
12 hours to remove the ethanol/water; 
(4) milling the dried granules to a uniform size using a Quadro Comill or 
Fitz type mill; 
(5) adding and blending croscarmellose sodium with the dried milled 
particles for about 5 to 30 minutes; 
(6) adding and blending magnesium stearate to the mixture containing the 
croscarmellose sodium with a V blender for about 1 to 5 minutes; and 
(7) compressing the lubricated granules mixture into a desired tablet form. 
Another example of the invention is the process wherein the active 
ingredient is 
N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-1'- 
yl)carbonyl]-2-(phenylmethyloxy)-ethyl]-2-amino-2-methylpropanamide 
methanesulfonate. 
In a subclass is the foregoing process further comprising the step of 
coating the tablet. Further exemplifying the invention is the foregoing 
process further comprising the steps of: 
(1) dry blending titanium dioxide (optionally mixed with talc) with 
hydroxypropyl methylcellulose and hydroxypropyl cellulose to form a dry 
powdered blend; 
(2) adding the dry powdered blend to water to form a slurry; 
(3) adding water to the slurry with stirring to form a suspension; and 
(4) applying the suspension to the tablets. 
An additional illustration of the present invention is a solid dosage form 
containing an active ingredient of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide, or a 
pharmaceutically acceptable salt thereof, in particular the 
methanesulfonate salt, wherein the dosage form is prepared by the process. 
The present invention further relates to a novel amorphous form of the 
compound 
N-[1(R)-[(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1' 
-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate. This amorphous form is produced directly as a result of 
the instant process of tablet formulation. 
The amorphous form of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-1'- 
yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate exhibits a lack of crystallinity. The lack of 
crystallinity was confirmed by X-ray analysis wherein he X-ray diffraction 
pattern showed an amorphous halo. 
The X-ray powder diffraction (XRPD) pattern was collected using a Phiulips 
APD3720 Automated Powder Diffraction instrument with copper K.alpha. 
radiation. Measurements were made from 2.degree. to 40.degree. (2 theta) 
with the sample maintained at ambient room temperature. 
In addition, examination of the amorphous form under microscopy showed no 
biorefringence. 
The amorphous form may be prepared by evaporating a concentrated solution 
of N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 
1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate in 25% aqueous ethanol (980 mg/ml) at 40.degree. C. to 
give a solid. 
Granulation is the process of adding a solvent, such as water or 
water/ethanol, to a powder mixture until granules are formed. The 
granulation step may be varied from 2 to 35 minutes, preferably 3 to 10 
minutes, most preferably 4 to 8 minutes. Preferably, the granules are 
dried using a fluid bed dryer or tray dryer. Milling of the dried granules 
is accomplished using a Quadro Comill or Fitz mill. The lubrication step 
is the process of adding lubricant to the mixture. The lubrication step 
may be varied from 30 seconds to 20 minutes, preferably about 1 minute. 
The disclosed process may be used to prepare solid dosage forms, 
particularly tablets or granules, for medicinal administration. 
The term "tablet," as used herein, is intended to encompass compressed 
pharmaceutical dosage formulations of all shapes and sizes, whether coated 
or uncoated. Substances which may be used for coating include 
hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), 
titanium dioxide, talc, sweeteners and colorants. 
The term "active ingredient," as used herein includes both the free base 
N-[1(R)-[(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1' 
-yl)carbonyl)-2-(phenyl-methyl-oxy)ethyl]-2-amino-2-methyl-propanamide, as 
well as the pharmaceutically acceptable salts thereof, in particular, 
N-[1(R)-f(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1' 
-yl)carbonyl]-2-(phenyl-methyl-oxy)ethyl]-2-amino-2-methyl-propanamide 
methanesulfonate and crystal forms thereof. A preferred crystal form for 
use in the present invention is that designated Form I. 
Preferred diluents include: lactose, microcrystalline cellulose, calcium 
phosphate(s), mannitol, powdered cellulose, pregelatinized starch and 
other suitable diluents (see, e.g., Remington's Pharmaceutical Sciences, 
18th Edition, 1990, p. 1635). Microcrystalline cellulose and calcium 
phosphate dibasic, are particularly preferred. Specifically, 
microcrystalline cellulose NF, especially Avicel PH101, the trademarked 
name for microcrystalline cellulose NF manufactured by FMC Corp. is 
preferred. 
Preferred binders include pregelatinized starch, hydroxypropyl 
methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone (PVP) and 
other known binders (see, e.g., Remington's Pharmaceutical Sciences, 18th 
Edition, 1990, pp. 1635-1636) and mixtures thereof. Most preferably, 
pregelatinized starch as employed as a binder. Specifically, starch 
pregelatinized NF 1500 manufactured by Colorcon Corporation is most 
preferred. 
The disintegrant may be one or more of several starches, clays, celluloses, 
algins, gums or crosslinked polymers known to those skilled in the art 
(See, e.g., Remington's Pharmaceutical Sciences, 18th Edition, 1990, p. 
1637) and mixtures thereof. Preferably, one or more of several modified 
starches or modified cellulose polymers, such as microcrystalline 
cellulose and croscarmellose sodium, are used. Croscarmellose sodium Type 
A, commercially available under the trade name "Ac-di-sol," is 
particularly preferred. 
Preferred lubricants include magnesium stearate, zinc stearate, calcium 
stearate, stearic acid, surface active agents such as sodium lauryl 
sulfate, magnesium lauryl sulfate, propylene glycol, sodium dodecane 
sulfonate, sodium oleate sulfonate and sodium laurate mixed with stearates 
and talc, sodium stearyl fumarate, hydrogenated vegetable oils, glyceryl 
palmitostearate, glyceryl behenate, sodium benzoate, mineral oil, talc and 
other known lubricants (see, e.g., Remington's Pharmaceutical Sciences, 
18th Edition, 1990, pp. 1636-1637), and mixtures thereof. An especially 
preferred lubricant is magnesium stearate. 
The active ingredient, 
N-[1(R)-[(1,2-di-hydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'- 
yl)carbonyl)-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide, may be 
prepared according to the methods disclosed in U.S. Pat. No. 5,536,716, 
PCT Patent Publication WO 94/13696 and the methods disclosed herein. 
The pharmaceutically acceptable salts of 
N-[1(R)-[(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1' 
-yl)carbonyl]-2-(phenyl-methyl-oxy)ethyl]-2-amino-2-methylpropanamide may 
be employed in the instant invention. Examples of pharmaceutically 
acceptable salts include the pharmaceutically acceptable acid addition 
salts, such as the salts derived from using inorganic and organic acids. 
Examples of such acids are hydrochloric, nitric, sulfuric, phosphoric, 
formic, acetic, trifluoroacetic, propionic, maleic, succinic, malonic, 
methane sulfonic and the like. 
The pharmaceutical compositions of the present invention comprise 0.1 to 
50% by weight of an active ingredient, 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide, or a 
pharmaceutically acceptable salt thereof, preferably 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate; 0 to 77% by weight of a binder/diluent; 0 to 77% by 
weight of a first diluent; 0 to 77% by weight of a second diluent; 0 to 6% 
by weight of a disintegrant; and 0 to 5% by weight of a lubricant. It will 
be appreciated by one skilled in the art that the sum of the proportions 
of the active ingredient, the binder/diluent, the first diluent, the 
second diluent, the disintegrant, and the lubricant are not greater than 
100% by weight. 
More specifically, the binder/diluent is selected from hydroxy-propyl 
methylcellulose, hydroxypropyl cellulose, pregelatinized starch or 
polyvinylpyrrolidone; the first and second diluents are independently 
selected from lactose, microcrystalline cellulose, calcium phosphate 
dibasic, mannitol, powdered cellulose or pregelatinized starch; the 
disintegrant is selected from microcrystalline or croscarmellose sodium; 
and the lubricant is selected from magnesium stearate, calcium stearate, 
steric acid or a surface active agent. 
In a specific embodiment, the binder/diluent is pregelatinized starch; the 
first diluent is microcrystalline cellulose; the second diluent is calcium 
phosphate dibasic; the disintegrant is croscarmellose sodium; and the 
lubricant is magnesium stearate. 
The pharmaceutical compositions of the present invention are preferably in 
the form of tablets. The tablets may be, for example, from 50 mg to 1.0 g 
in net weight, preferably 100 to 800 mg net weight, more preferably 100 to 
400 mg net weight. 
Preferred pharmaceutical compositions comprise about 1 to 30% by weight of 
the active ingredient; about 20 to 40% by weight of pregelatinized starch; 
about 10 to 20% by weight of microcrystalline cellulose; about 20 to 50% 
by weight of calcium phosphate dibasic; about 5 to 15% by weight of 
croscarmellose sodium; and about 0.05 to 5% by weight of magnesium 
stearate. 
It will be appreciated by one skilled in the art that the sum of the above 
proportions of the active ingredient, pregelatinized starch, 
microcrystalline cellulose, calcium phosphate dibasic, croscarmellose 
sodium, and magnesium stearate are not greater than 100% by weight. 
More preferred pharmaceutical compositions in accordance with the present 
invention include those comprising the noted ingredients: 
(1) about 1 to 2% by weight of the active ingredient; about 25 to 35% by 
weight of pregelatinized starch; about 10 to 20% by weight of 
microcrystalline cellulose; about 45-55% by weight of calcium phosphate 
dibasic; about 4 to 8% by weight of croscarmellose sodium; and about 0.1 
to 1% by weight of magnesium stearate. 
(2) about 5 to 10% by weight of the active ingredient, about 25 to 35% by 
weight of pregelatinized starch; about 10 to 20% by weight of 
microcrystalline cellulose; about 40 to 50% by weight of calcium phosphate 
dibasic; about 4 to 8% by weight of croscarmellose sodium; and about 0.1 
to 1% by weight of magnesium stearate. 
(3) about 25 to 35% by weight of the active ingredient, about 15 to 25% by 
weight of pregelatinized starch; about 10 to 20% by weight of 
microcrystalline cellulose; about 15-25% by weight of calcium phosphate 
dibasic; about 10 to 20% by weight of croscarmellose sodium; and about 0.1 
to 1% by weight of magnesium stearate. 
It will be appreciated by one skilled in the art that the sum of the above 
proportions of the active ingredient, pregelatinized starch, 
microcrystalline cellulose, calcium phosphate dibasic, croscarmellose 
sodium, and magnesium stearate are not greater than 100% by weight. 
Especially referred pharmaceutical compositions as envisioned for 
commercial development are as follows: 
Tablets of 1.0 mg potency free base: 
about 1.18% by weight of active ingredient as the methanesulfonate salt; 
about 30.0% by weight of pregelatinized starch; about 15.0% by weight of 
microcrystalline cellulose; about 47.3% by weight of calcium phosphate 
dibasic; about 6.0% by weight of croscarmellose sodium; and about 0.5% by 
weight of magnesium stearate. This composition comprises about 1.2 mg of 
active ingredient as the methanesulfonate salt; about 30 mg of 
pregelatinized starch; about 15 mg of microcrystalline cellulose; about 
47.3 mg of calcium phosphate dibasic; about 6.0 mg of croscarmellose 
sodium; and about 0.5 mg of magnesium stearate per dosage unit. 
Optionally, the 1.0 mg potency tablet may be coated with a coating 
comprising about 0.8% by weight of hydroxypropyl methylcellulose; about 
0.8% by weight of hydroxypropyl cellulose; about 0.32% by weight of 
titanium dioxide; and about 0.08% by weight of talc (as a percentage of 
the core tablet weight). 
Tablets of 5.0 mg potency free base: 
about 1.48% by weight of active ingredient as the methanesulfonate salt; 
about 30.0% by weight of pregelatinized starch; about 15.0% by weight of 
microcrystalline cellulose; about 47.0% by weight of calcium phosphate 
dibasic; about 6.0% by weight of croscarmellose sodium; and about 0.5% by 
weight of magnesium stearate. This composition comprises about 6 mg of 
active ingredient as the methanesulfonate salt; about 120 mg of 
pregelatinized starch; about 60 mg of microcrystalline cellulose; about 
188 mg of calcium phosphate dibasic; about 24 mg of croscarmellose sodium; 
and about 2 mg of magnesium stearate per dosage unit. 
Optionally, the 5.0 mg potency tablet may be coated with a coating 
comprising about 0.8% by weight of hydroxypropyl methylcellulose; about 
0.8% by weight of hydroxypropyl cellulose; about 0.32% by weight of 
titanium dioxide; and about 0.08% by weight of talc (as a percentage of 
the core tablet weight). 
Tablets of 25 mg potency free base: 
about 7.39% by weight of active ingredient as the methanesulfonate salt; 
about 282% by weight of pregelatinized starch; about 14.2% by weight of 
microcrystalline cellulose; about 43.6% by weight of calcium phosphate 
dibasic; about 6.0% by weight of croscarmellose sodium; and about 0.5% by 
weight of magnesium stearate. This composition comprises about 30 mg of 
active ingredient as the methanesulfonate salt; about 113 mg of 
pregelatinized starch; about 57 mg of microcrystalline cellulose; about 
174 mg of calcium phosphate dibasic; about 24 mg of croscarmellose sodium; 
and about 2 mg of magnesium stearate per dosage unit. 
Optionally, the 25 mg potency tablet may be coated with a coating 
comprising about 0.8% by weight of hydroxypropyl methylcellulose; about 
0.8% by weight of hydroxypropyl cellulose; about 0.32% by weight of 
titanium dioxide; and about 0.08% by weight of talc (as a percentage of 
the core tablet weight). 
Tablets of 100 mg potency free base: 
about 29.5% by weight of active ingredient as the methanesulfonate salt; 
about 19.5% by weight of pregelatinized starch; about 15.0% by weight of 
microcrystalline cellulose; about 20.4% by weight of calcium phosphate 
dibasic; about 15.0% by weight of croscarmellose sodium; and about 0.5%o 
by weight of magnesium stearate. This composition comprises about 118 mg 
of active ingredient as the methanesulfonate salt; about 78 mg of 
pregelatinized starch; about 60 mg of microcrystalline cellulose; about 82 
mg of calcium phosphate dibasic; about 60 mg of croscarmellose sodium; and 
about 2 mg of magnesium stearate per dosage unit. 
Optionally, the 100 mg potency tablet may be coated with a coating 
comprising about 0.8% by weight of hydroxypropyl methylcellulose; about 
0.8% by weight of hydroxypropyl cellulose; about 0.32% by weight of 
titanium dioxide; and about 0.08% by weight of talc (as a percentage of 
the core tablet weight). 
The tablets of the 1.0 mg potency are preferably formulated in an 100 mg 
tablet by using 30 .mu.l of a solution of 25% ethanol/75% water per 
tablet. The tablets of the 5.0 mg potency are preferably formulated in an 
400 mg tablet by using 120 .mu.l of a solution of 25% ethanol/75% water 
per tablet. The tablets of the 25 mg potency are preferably formulated in 
an 400 mg tablet by using 120 .mu.l of a solution of 25% ethanol/75% water 
per tablet. The tablets of the 100 mg potency are preferably formulated in 
an 400 mg tablet by using 120 .mu.l of a solution of 25% ethanol/75% water 
per tablet. 
In a particularly preferred embodiment, the tablet formulations of the 
instant invention are coated. In the pharmaceutical compositions 
envisioned for commercial development described above, the tablets of 1.0 
mg, 5.0 mg, 25 mg and 100 mg potency free base are coated with about 0.8% 
by weight of hydroxypropyl methylcellulose; about 0.8% by weight of 
hydroxypropyl cellulose; about 0.32% by weight titanium dioxide; and about 
0.08% by weight of purified talc. 
In the most preferred embodiment, the active ingredient in the 
above-described pharmaceutical compositions is 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl)-2-amino-2-methylpropanamide 
methanesulfonate. 
The compositions of the present invention are in a form for oral 
administration and may take the form of tablets, capsules, granules, 
powders, tablets or granules for buccal administration, or liquid 
preparations such as suspensions. Granules and powders may be ingested 
directly, or dispersed in water or other suitable vehicle prior to 
administration. Capsules may be of the hard or soft gelatin type, 
including soft gelatin capsules. 
The pharmaceutical compositions of the present invention may also contain 
other excepients conventional in the art such as flavorings, sweeteners, 
and the like. Suitable flavorings include for example fruit flavors or 
natural or synthetic mint or peppermint flavors. Suitable sweeteners 
include for example sugar, saccharin or aspartame. 
The utility of the active ingredient of the formulation of the present 
invention as growth hormone secretagogues may be demonstrated by 
methodology known in the art, such as an assay described by Smith, et al., 
Science, 260, 1640-1643 (1993) (see text of FIG. 2 therein). In 
particular, the active ingredient used in the formulation the present 
invention had activity as a growth hormone secretagogue in the 
aforementioned assay. Such a result is indicative of the activity of the 
formulation of the present invention as a growth hormone secretagogue. 
The formulations of the present invention may be administered to animals, 
including man, to release growth hormone in vivo. For example, the 
formulations can be administered to commercially important animals such as 
swine, cattle, sheep and the like to accelerate and increase their rate 
and extent of growth, to improve feed efficiency and to increase milk 
production in such animals. In addition, these formulations can be 
administered to humans in vivo as a diagnostic tool to directly determine 
whether the pituitary is capable of releasing growth hormone. For example, 
the formulation of the present invention can be administered in vivo to 
children. Serum samples taken before and after such administration can be 
assayed for growth hormone. Comparison of the amounts of growth hormone in 
each of these samples would be a means for directly determining the 
ability of the patient's pituitary to release growth hormone. 
Accordingly, the present invention includes within its scope pharmaceutical 
compositions comprising, as an active ingredient, the compound 
N-[1(R)-[(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1' 
-yl)carbonyl]-2-(phenyl-methyl-oxy)ethyl]-2-amino-2-methyl-propanamide in 
association with a pharmaceutical carrier or diluent. Optionally, the 
active ingredient of the pharmaceutical compositions may comprise an 
anabolic agent in addition to the compound 
N-[1(R)-[(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1' 
-yl)carbonyl]-2-(phenyl-methyl-oxy)ethyl]-2-amino-2-methyl-propanamide or 
another composition which exhibits a different activity, e.g., an 
antibiotic growth permittant or an agent to treat osteoporosis or in 
combination with a corticosteroid to minimize the catabolic side effects 
or with other pharmaceutically active materials wherein the combination 
enhances efficacy and minimizes side effects. 
Growth promoting and anabolic agents include, but are not limited to TRH, 
diethylstilbesterol, amino acids, estrogens, .beta.-agonists, 
theophylline, anabolic steroids, enkephalins, E series prostaglandins, 
compounds disclosed in U.S. Pat. No. 3,239,345, e.g., zeranol, and 
compounds disclosed in U.S. Pat. No. 4,036,979, e.g., sulbenox or peptides 
disclosed in U.S. Pat. No. 4,411,890. 
A still further use of the formulations of this invention is in combination 
with other growth hormone secretagogues such as the growth hormone 
releasing peptides GHRP-6, GHRP-1 as described in U.S. Pat. No. 4,411,890 
and publications WO 89/07110, WO 89/07111 and B-HT920 as well as hexarelin 
and GHRP-2 as described in WO 93/04081 or growth hormone releasing hormone 
(GHRH, also designated GRF) and its analogs or growth hormone and its 
analogs or somatomedins including IGF-1 and IGF-2 or .alpha.-adrenergic 
agonists such as clonidine or serotonin 5HTID agonists such as sumitriptan 
or agents which inhibit somatostatin or its release such as physostigmine 
and pyridostigmine. In particular, the formulations of this invention may 
be used in combination with growth hormone releasing factor, an analog of 
growth hormone releasing factor, IGF-1, or IGF-2. For example, a 
formulation of the present invention may be used in combination with IGF-1 
for the treatment or prevention of obesity. In addition, a formulation of 
this invention may be employed in conjunction with retinoic acid to 
improve the condition of musculature and skin that results from intrinsic 
aging. 
As is well known to those skilled in the art, the known and potential uses 
of growth hormone are varied and multitudinous. The administration of the 
formulations of this invention for purposes of stimulating the release of 
endogenous growth hormone can have the same effects or uses as growth 
hormone itself. These varied uses of the present formulations thus may be 
summarized as follows: stimulating growth hormone release in elderly 
humans; treating growth hormone deficient adults; prevention of catabolic 
side effects of glucocorticoids; treatment of osteoporosis; stimulation of 
the immune system, acceleration of wound healing; accelerating bone 
fracture repair; treatment of growth retardation; treating acute or 
chronic renal failure or insufficiency; treatment of physiological short 
stature, including growth hormone deficient children; treating short 
stature associated with chronic illness; treatment of obesity and growth 
retardation associated with obesity; treating growth retardation 
associated with Prader-Willi syndrome and Turner's syndrome; accelerating 
the recovery and reducing hospitalization of burn patients or following 
major surgery such as gastrointestinal surgery; treatment of intrauterine 
growth retardation, and skeletal dysplasia, treatment of peripheral 
neuropathies; replacement of growth hormone in stressed patients; 
treatment of osteochondrody-splasias, Noonans syndrome, schizophrenia, 
depression, Alzheimer's disease, delayed wound healing, and psychosocial 
deprivation; treatment of pulmonary dysfunction and ventilator dependency; 
attenuation of protein catabolic response after a major operation; 
treating malabsorption syndromes; reducing cachexia and protein loss due 
to chronic illness such as cancer or AIDS; accelerating weight gain and 
protein accretion in patients on TPN (total parenteral nutrition); 
treatment of hyperinsulinemia including nesidioblastosis; adjuvant 
treatment for ovulation induction and to prevent and treat gastric and 
duodenal ulcers; to stimulate thymic development and prevent the 
age-related decline of thymic function; adjunctive therapy for patients on 
chronic hemodialysis; treatment of immunosuppressed patients and to 
enhance antibody response following vaccination; increasing the total 
lymphocyte count of a human, in particular, increasing the T.sub.4 
/T.sub.8 -cell ratio in a human with a depressed T.sub.4 /T.sub.8 -cell 
ratio resulting, for example, from physical trauma, such as closed head 
injury, or from infection, such as bacterial or viral infection, 
especially infection with the human immunodeficiency virus; improvement in 
muscle strength, mobility, maintenance of skin thickness, metabolic 
homeostasis, renal hemeostasis in the frail elderly; stimulation of 
osteoblasts, bone remodelling, and cartilage growth; stimulation of the 
immune system in companion animals and treatment of disorders of aging in 
companion animals; growth promotant in livestock; and stimulation of wool 
growth in sheep. Further, the instant compounds are useful for increasing 
feed efficiency, promoting growth, increasing milk production and 
improving the carcass quality of livestock. Likewise, the instant 
formulations are useful in a method of treatment of diseases or conditions 
which are benefited by the anabolic effects of enhanced growth hormone 
levels. 
In particular, the instant formulations are useful in the prevention or 
treatment of a condition selected from the group consisting of: 
osteoporosis; catabolic illness; immune deficiency, including that in 
individuals with a depressed T.sub.4 /T.sub.8 cell ratio; hip fracture; 
musculoskeletal impairment in the elderly; growth hormone deficiency in 
adults or in children; obesity; cachexia and protein loss due to chronic 
illness such as AIDS or cancer; and treating patients recovering from 
major surgery, wounds or burns, in a patient in need thereof. 
In addition, the instant formulations may be useful in the treatment of 
illnesses induced or facilitated by corticotropin releasing factor or 
stress- and anxiety-related disorders, including stress-induced depression 
and headache, abdominal bowel syndrome, immune suppression, HIV 
infections, Alzheimer's disease, gastrointestinal disease, anorexia 
nervosa, hemorrhagic stress, drug and alcohol withdrawal symptoms, drug 
addiction, and fertility problems. 
It will be known to those skilled in the art that there are numerous 
compounds now being used in an effort to treat the diseases or therapeutic 
indications enumerated above. Combinations of these therapeutic agents 
some of which have also been mentioned above in and with the formulations 
of this invention will bring additional, complementary, and often 
synergistic properties to enhance the growth promotant, anabolic and 
desirable properties of these various therapeutic agents. In these 
combinations, the therapeutic agents and the active ingredient in the 
formulations of this invention may be independently present in dose ranges 
from one one-hundredth to one times the dose levels which are effective 
when these compounds and active ingredients are used singly. 
Combined therapy to inhibit bone resorption, prevent osteoporosis and 
enhance the healing of bone fractures can be illustrated by combinations 
of bisphosphonates and the formulations of this invention. The use of 
bisphosphonates for these utilities has been reviewed, for example, by 
Hamdy, N. A. T., Role of Bisphosphonates in Metabolic Bone Diseases, 
Trends in Endocrinol. Metab., 4, 19-25 (1993). Bisphosphonates with these 
utilities include alendronate, tiludronate, dimethyl-APD, risedronate, 
etidronate, YM-175, clodronate, pamidronate, and BM-210995. According to 
their potency, oral daily dosage levels of the bisphosphonate of between 
0.1 mg and 5 g and daily dosage levels of the active ingredient of the 
formulation of this invention of between 0.01 mg/kg to 20 mg/kg of body 
weight are administered to patients to obtain effective treatment of 
osteoporosis. 
In the case of alendronate daily oral dosage levels of 0.1 mg to 50 mg are 
combined for effective osteoporosis therapy with 0.01 mg/kg to 20 mg/kg of 
the active ingredient employed in the formulation of this invention. 
Osteoporosis and other bone disorders may also be treated with the 
formulations of this invention in combination with calcitonin, estrogens, 
raloxifene and calcium supplements such as calcium citrate. 
Anabolic effects especially in the treatment of geriatric male patients are 
obtained with formulations of this invention in combination with anabolic 
steroids such as oxymetholone, methyltesterone, fluoxymesterone and 
stanozolol. 
The pharmaceutical tablet compositions of the present invention may also 
contain one or more additional formulation ingredients selected from a 
wide variety of excipients (also referred to as "additives") known in the 
pharmaceutical formulation art. According to the desired properties of the 
tablet, any number of additives may be selected, alone or in combination, 
based upon their known uses in preparing tablet compositions. Such 
additives include, but are not limited to, diluents, binders, compression 
aids, disintegrants, lubricants, flavors, flavor enhancers, sweeteners and 
preservatives. Due to the bitter taste of the active ingredient, the 
inclusion of a sweetener may be desired. 
The dosage of active ingredient in the compositions of this invention may 
be varied; however, it is necessary that the amount of the active 
ingredient be such that a suitable dosage form is obtained. The selected 
dosage depends upon the desired therapeutic effect, on the route -of 
administration, and on the duration of the treatment. Generally, dosage 
levels of between 0.0001 to 10 mg/kg. of body weight daily are 
administered to patients and animals, e.g., mammals, to obtain effective 
release of growth hormone. Preferably, the dosage level will be about 
0.001 to about 25 mg/kg per day; more preferably about 0.01 to about 10 
mg/kg per day. 
Methods for preparing the formulations of the present invention, as well as 
the active ingredient are illustrated in the following Examples. The 
following examples are given for the purpose of illustrating the present 
invention and shall not be construed as being limitations on the scope or 
spirit of the instant invention.

EXAMPLE 1 
N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl 
)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
Step A: 
1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdine]hydrochloride 
To a solution of 1.20 g (5.8 mmol) of 
1'-methyl-1,2-dihydro-spiro[3H-indole-3,4'-piperdine] (prepared as 
described by H. Ong, et al., J. Med. Chem., 23, 981-986 (1983)) in 20 mL 
of dry dichloromethane at 0.degree. C. was added triethylamine (0.90 mL; 
6.4 mmol) and methanesulfonyl chloride (0.49 mL; 6.35 mmol) and stirred 
for 30 min. The reaction mixture was poured into 15 mL of saturated 
aqueous sodium bicarbonate solution and extracted with dichloromethane 
(2.times.10 mL). The combined organics were washed with brine (20 mL), 
dried over anhydrous potassium carbonate, filtered and the solvent removed 
under reduced pressure to yield 1.44 g of the methanesulfonamide 
derivative as pale yellow oil which was used without purification. 
To a solution of above crude product in 20 mL of dry 1,2-dichloroethane at 
0.degree. C. was added 1.0 mL (9.30 mmol) of 1-chloroethyl chloroformate, 
and then stirred at RT for 30 min and finally at reflux for 1 h. The 
reaction mixture was concentrated to approximately one third of the volume 
and then diluted with 20 mL of dry methanol and refluxed for 1.5 h. The 
reaction was cooled to RT and concentrated to approximately one half of 
the volume. The precipitate was filtered and washed with a small volume of 
cold methanol. This yielded 1.0 g of the piperidine HCl salt as a white 
solid. The filtrate was concentrated and a small volume of methanol was 
added followed by ether. The precipitated material was once again 
filtered, washed with cold methanol, and dried. This gave an additional 
0.49 g of the desired product. Total yield 1.49 g (70%). 
.sup.1 H NMR (CDCl.sub.3, 200 MHz) .delta. 7.43-7.20 (m, 3H), 7.10 (dd, 
1H), 3.98 (bs, 2H), 3.55-3.40 (bd, 2H), 3.35-3.10 (m, 2H), 2.99 (s, 3H), 
2.15 (t, 2H), 2.00 (t, 2H). 
Step B: 
N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-[(1,1-dimethylethoxy)carbonyl]amino 
-2-methyl-propanamide 
To 0.35 g (1.15 mmol) of 
(2R)-2-[(1,1-dimethylethoxy)carbonyl]amino-3-[2-(phenylmethyloxy)ethyl]-1- 
propanoic acid in 13 mL of dichloromethane was added 
1,2-dihydro-1-methanesulfonylspiro-[3H-indole-3,4'-piperdine) 
hydrochloride (0.325 g; 1.07 mmol), 0.18 -mL (1.63 mmol) of 
N-methylmorpholine, 0.159 g (1.18 mmol) of 1-hydroxybenztriazole(HOBT) and 
stirred for 15 min. EDC (0.31 g; 1.62 mol) was added and stirring was 
continued for 1 h. An additional 60 .mu.L of N-methylmorpholine was added 
and stirred for 45 min. The reaction mixture was poured into 5 mL of water 
and the organic layer was separated. The organic layer was washed with 5 
mL of 0.5N aqueous hydrochloric acid and 5 mL of saturated aqueous sodium 
bicarbonate solution. The combined organics were dried over anhydrous 
magnesium sulfate, and concentrated to yield 0.627 g of the product as a 
yellow foam which was used without purification. 
To a 0.627 g (1.07 mmol) of the above product in 5 mL of dichloromethane 
was added 1.0 mL of trifluoroacetic acid and stirred at RT for 75 min. An 
additional 1.00 mL of trifluoroacetic acid was added and stirred for 10 
min. The reaction mixture was concentrated, diluted with 5.0 mL of 
dichloromethane and carefully basified by pouring into 10 mL of 10% 
aqueous sodium carbonate solution. The organic layer was separated and the 
aqueous layer was further extracted with 2.times.15 mL of dichloromethane. 
The combined organics were washed with 5 mL of water, dried over potassium 
carbonate, filtered and concentrated to give the 0.486 g of the amine as a 
light yellow foam which was used without purification. 
To 0.486 g (1.01 mmol) of the amine and 10 mL of dichloromethane was added 
0.26 g (1.28 mmol) of 
2-[(1,1-dimethylethoxy)carbonyl]amino-2-methyl-propanoic acid, 0.173 g 
(1.28 mmol) of 1-hydroxybenztriazole (HOBT) and EDC (0.245 g; 1.28 mol) 
and stirried at RT overnight. The reaction mixture was poured into 5.0 mL 
of water and the organic layer was separated. The aqueous layer was back 
extracted with 5 mL of dichloromethane. The combined organics were washed 
with 5.0 mL of 0.5N aqueous hydrochloric acid, 5 mL of saturated aqueous 
sodium bicarbonate solution dried over anhydrous magnesium sulfate, and 
concentrated to yield 0.751 g of the crude product as a yellow foam. A 
solution of this crude product in dichloromethane was chromatographed on 
25 g of silica gel and eluted first with hexanes/acetone/dichloromethane 
(70/25/5) and then with hexanes/acetone/dichloromethane (65/30/5). This 
gave 0.63 g of the title compound as a white solid. 
.sup.1 H NMR (CDCl.sub.3, 400 MHz) Compound exists as a 3:2 mixture of 
rotamers .delta. 7.40-7.10 (m, 6H), 7.06 (d, 1/3H), 7.02 (t, 1/3H), 6.90 
(t, 1/3H), 6.55 (d, 1/3H), 5.15 (m, 1H), 4.95 (bs, 1H), 4.63 (bd, 1/3H), 
4.574.40 (m, 2 2/3 H), 4.10 (bd, 1/3H), 4.00 (bd, 1/3H), 3.82 (t, 1H), 
3.78-3.62 (m, 2H), 3.60-3.50 (m, 1H), 3.04 (q, 1H), 2.87 (s, 1H), 2.86 (s, 
2H), 2.80-2.60 (m, 1H), 1.90 (bs, 1H), 2.85-2.75 (m, 1H), 1.82-1.60 (m, 
3H), 1.55-1.45 (m, 1H), 1.45 (s, 4H), 1.42 (s, 2H), 1.39 (s, 9H). 
Step C: 
N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperidin]-1'- 
yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
hydrochloride 
To 0.637 g (0.101 mmol) of the intermediate from Step B in 5 mL of 
dichloromethane was added 2.5 mL of trifluoroacetic acid and stirred at RT 
for 30 min. The reaction mixture was concentrated to an oil, taken up in 
10 mL of ethyl acetate and washed with 8 mL of 10% aqueous sodium 
carbonate solution. The aqueous layer was further extracted with 5 mL of 
ethyl acetate. The combined organics were washed with 10 mL of water, 
dried over magnesium sulfate, filtered and concentrated to give the 0.512 
g of the free base as a white foam. 
To 0.512 g of the free base in 5 mL of ethyl acetate at 0.degree. C. was 
added 0.2 mL of saturated hydrochloric acid in ethyl acetate and stirred 
for 1.5 h. The white precipitate was filtered under nitrogen, washed with 
ether, and dried to give 0.50 g of the title compound as a white solid. 
.sup.1 H NMR (400 MHz, CD.sub.3 OD) Compound exists as 3:2 mixture of 
rotamers. .delta. 7.40-7.28 (m, 4H), 7.25-7.17 (m, 2H), 7.08 (t, 1/3H), 
7.00 (t, 1/3H), 6.80 (d, 1/3H), 5.16 (ddd, 1H), 4.60-4.42 (m, 3H), 4.05 
(t, 1H), 3.90 (bs, 2H), 3.83-3.70 (m, 2H), 3.30-3.15 (m, 1H), 2.97 (s, 
1H), 2.95 (s, 2H), 2.90-2.78 (m, 1H), 1.96 (t, 1/3H), 1.85-1.65 (m, 4H), 
1.63 (s, 2H), 1.60 (s, 4H). 
EXAMPLE 2 
N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl 
) carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
Step A: 
(2R)-[[[-2-(1,1-dimethylethoxy)carbonyl]amino]-2,2-dimethyl-1-oxoethyl]ami 
no-2-(phenylmethoxy)ethyl]-1-propanoic acid allyl ester 
Prepared from 
(2R)-2-[(1,1-dimethylethoxy)carbonyl]-amino-3-(phenylmethyloxy)ethyl-propa 
noic acid and allyl alcohol by carrying out the coupling reaction in 
CH.sub.2 Cl.sub.2 in the presence of EDC and DMAP. 
.sup.1 H NMR (400 MHz, CDCl.sub.3) .delta. 7.25 (s, 5H), 5.8 (m, 1H), 5.2 
(dd, 2H), 5.0 (bs, 1H), 4.7 (m, 1H), 4.6 (m, 2H), 4.4 (dd, 2H), 3.9 (dd, 
1H), 3.6 (dd, 1H), 1.45 (d, 6H), 1.39 (s, 9H). 
Step B: 
(2R)-[[[-2-(1,1-dimethylethoxy)carbonyl]amino]-2,2-dimethyl-1-oxoethyl] 
amino-2-(phenylmethyloxy)ethyl)-1-propanoic acid 
To a stirred solution of the crude intermediate obtained in Step A (6.7 g, 
15.9 mmol), tetrakis (triphenylphosphine)-palladium (1.8 g, 0.1 eq) and, 
triphenyl phosphine (1.25 g, 0.3 eq) was added a solution of 
potassium-2-ethyl hexanoate (35 mL, 0.5M solution in EtOAc). The reaction 
mixture was stirred at room temperature under nitrogen atmosphere for 1 h 
and then diluted with ether (100 mL) and poured into ice-water. The 
organic layer was seperated and the aqueous fraction was acidified with 
citric acid (20%), then extracted with EtOAc. The EtOAc extracts were 
washed with brine, dried over magnesium sulfate, filtered and evaporated 
to give the title compound as a solid. 
.sup.1 H NMR (400Hz, CD.sub.3 OD) .delta. 7.3 (s, 5H), 4.7 (m, 1H), 4.5 (s, 
2H), 4.0 (m, 1H), 3.6 (m, 1H), 1.4 (d, 6H), 1.3 (s, 9H). 
Step C: 
N-[(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl 
)carbonyl]-2-(phenylmethyloxy)ethyl]-2-[(1,1-dimethyl-ethoxy)carbonyl]amino 
-2-methyl-propanamide 
To a solution of 1.0 g (3.44 mmol) of 
1-methanesulfonylspiro[indoline-3,4'-piperidine] hydrochloride, 1.44 g 
(3.78 mmol) of 
(2R)-[[-2-(1,1-dimethylethoxy)carbonyl)amino]-2,2-dimethyl-1-oxo-ethyl]-am 
ino-2-(phenylmethyloxy)ethyl)-1-propanoic acid, N-methyl morpholine (0.58 
mL; 5.20 mmol), and 1-hydroxybenztriazole (HOBT) (0.58 g; 3.78 mmol), in 
50 mL of dichloromethane was added EDC (1.03 g; 5.20 mmol) and stirred at 
RT for 16 h. The reaction mixture was diluted with an additional 50 mL of 
dichloromethane and washed with aqueous sodium bicarbonate solution (50 
mL), dried over anhydrous magnesium sulfate, filtered, and concentrated. 
Flash chromatography (50 g silica gel) of the crude oily residue gave 
2.148 g (90%) of the desired material as a colorless foam. 
.sup.1 H NMR (CDCl.sub.3, 400 MHz) Compound exists as a 3:2 mixture of 
rotamers .delta. 7.40-7.10 (m, 6H), 7.06 (d, 1/3H), 7.02 (t, 1/3H), 6.90 
(t, 1/3H), 6.55 (d, 1/3H), 5.15 (m, 1H), 4.95 (bs, 1H), 4.63 (bd, 1/3H), 
4.574.40 (m, 2 2/3 H), 4.10 (bd, 1/3H), 4.00 (bd, 1/3H), 3.82 (t, 1H), 
3.78-3.62 (m, 2H), 3.60-3.50 (m, 1H), 3.04 (q, 1H), 2.87 (s, 1H), 2.86 (s, 
2H), 2.80-2.60 (m, 1H), 1.90 (bs, 1H), 2.85-2.75 (m, 1H), 1.82-1.60 (m, 
3H), 1.55-1.45 (m, 1H), 1.45 (s, 4H), 1.42 (s, 2H), 1.39 (s, 9H). 
Step D: 
N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
hydrochloride 
To a solution of 2.148 g (3.41 mmol) of the intermediate from Step C in 10 
mL of dichloromethane was added 5 mL of trifluoroacetic acid and stirred 
for 1 h. The reaction mixture was concentrated and basified with 100 mL of 
5% aqueous sodium carbonate solution and extracted with dichloromethane 
(3.times.50 mL). The combined organics were washed with brine (50 mL), 
dried over anhydrous potassium carbonate, filtered, and concentrated to 
yield a colorless foam. To a solution of the foam in 25 mL of ethyl 
acetate at 0.degree. C. was added 4 mL of 1 M solution of hydrochloric 
acid in ethyl acetate. The precipitate was filtered and washed first with 
ethyl acetate and then with ethyl acetate-ether (1:1), dried to yield 1.79 
g (93%) of the title compound as a colorless solid. 
.sup.1 H NMR (400 MHz, CD.sub.3 OD) Compound exists as 3:2 mixture of 
rotamers. .delta. 7.40-7.28 (m, 4H), 7.25-7.17 (m, 2H), 7.08 (t, 1/3H), 7) 
(t, 1/3H), 6.80 (d, 1/3H), 5.16 (ddd, 1H), 4.604.42 (m, 3H), 4.05 (1H), 
3.90 (bs, 2H), 3.83-3.70 (m, 2H), 3.30-3.15 (m, 1H0, 2.97 (s, 1H), 2.95 
(s, 2H), 2.90-2.78 (m, 1H), 1.96 (t, 1/3H), 1.85-1.65 (m, 4H), 1.63 (s, 
2H), 1.60 (s, 4H). 
EXAMPLE 3 
N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl 
)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide mesylate 
This compound was prepared by the treating the free base obtained in 
Example 5, Step D, with methane sulfonic acid. The title compound was 
obtained by recrystallizing it from ethyl acetate-ethanol-water. 
m.p.=166.degree.-168.degree. C. 
EXAMPLE 4 
##STR2## 
Isonipecotic acid-N-benzyl carbamate (3) 
______________________________________ 
Materials: 
______________________________________ 
Isonipecotic acid (2) T.C.I. 
4.02 kg (31.1 mol) 
Benzyl chloroformate (Schweitzerhall) 
6.91 kg (40.5 mol) 
K.sub.2 CO.sub.3 10.1 kg (72.9 mol) 
Water 40.2 L 
______________________________________ 
Isonipecotic acid (2) and K.sub.2 CO.sub.3 were dissolved in 40.2 L of 
water in a 100 L 4 neck flask with mechanical stirring under N.sub.2 and 
the solution was cooled to 10.degree. C. Benzyl chlorofornate was added, 
maintaining the temperature between 9 and 14.degree. C., and the mixture 
was warmed up to 22.degree. C. after the addition was complete and aged 
for 58 h. The addition was completed in 4 h at which point the pH was 9.0. 
After aging for 58 h there was no change in the pH. 
The reaction mixture was transferred to a 200 L extractor and washed with 
3.times.13 kg (15 L) of I and 1.times.12 L of EtOAc. The aqueous layer 
was extracted with 8 L of toluene. After the washes the benzyl alcohol 
content was reduced from 3.8% to 1.4% by HPLC analysis. HPLC analytical: 
Dupont Zorbax 25 cm RXC8 column with 1.5 ml/min flow and detection at 254 
nm; isocratic mixture with 35% MeCN, 65% of 0.1% aqueous H.sub.3 PO.sub.4 
; retention times: 3=6.9 min, benzyl alcohol=3.3 min, toluene=17.3 min. 
The aqueous phase was acidified with 37% aqueous HCl to pH 1.8. Carbon 
dioxide was evolved during the addition of HCl, but gas evolution was 
easily controlled. The addition of HCl took &lt;1 h and required 10 L of 
conc. HCl. The aqueous phase was extracted with 3.times.6.6 L of toluene. 
The toluene extracts were dried with 2 kg of sodium sulfate and filtered 
through a pad of Solka-floc.TM.. The combined filtrates weighed 17.8 kg. 
The crude yield of carbamate 3 was 7.89 kg (97%) (as obtained by 
evaporation of weighed aliquots of the filtrates to dryness). The 
filtrates were transferred through a 10.mu. inline filter to a 100 L 
flask. The extracts were concentrated at 10 mbar at &lt;25.degree. C. to a 
volume of 18 L. The final concentration of carbamate 3 was 440 g/L. The 
concentration of the toluene filtrate served to azeotropically remove 
final traces of water (final KF=170 mg/L). The product was 99.1 area % 
pure with 0.9 area % benzyl alcohol as the only impurity. 
EXAMPLE 5 
##STR3## 
Isonipecotic acid chloride-N-benzyl carbamate (4) 
______________________________________ 
Materials: 
______________________________________ 
Isonipecotic acid N-benzyl carbamate 
7.89 kg (30.0 mol) in 
in toluene. (MW = 263.30) 
17.9 L 
Oxalyl chloride (MW = 126.93) 
3.94 kg (31.0 mol) 
DMF (MW = 73.10) 10 mL 
Toluene 12 L 
______________________________________ 
To the toluene solution of benzyl carbamate 3 from the preceding step was 
added 5 mL of DMF and 10 L of toluene. The oxalyl chloride was added over 
a period of 20 min. The reaction mixture was aged for 16 h at 18.degree. 
C. under a slow stream of nitrogen. HPLC analysis of the reaction mixture 
showed that 1.3% of the carboxylic acid 3 still remained unreacted. The 
reaction mixture was warmed to 26.degree. C., and 5 mL of DMF were added. 
The mixture was aged for 2.5 h. A 1.0 mL aliquot of the reaction mixture 
was quenched with 5.0 mL of tert-butylamine and analyzed after evaporation 
by HPLC: 25 cm Dupont Zorbax RXC8 column at 50.degree. C. with 1 mL/min 
flow and detection at 220 nm; isocratic 42% MeCN, 58% of 0.1% aqueous 
H.sub.3 PO.sub.4. This method showed that &lt;0.05% of the acid 3 remained 
(as judged by A) and showed &gt;3 area % B (&gt;1 mol % (COCl).sub.2). 
##STR4## 
The mixture was concentrated at 10 mbar and a temperature of 20-25.degree. 
C. until 5 L of solvent had been removed. 
The typical HPLC profile of concentrated toluene solution after 
t-BuNH.sub.2 quench described above is as follows: 
______________________________________ 
Retention time (min) 
Area % Identity 
______________________________________ 
2.1 &lt;0.5% carboxylic acid 3 
7.8 &lt;0.5% benzyl chloride 
11.0 &gt;99% Cbz-t-butylcarboxamide A 
12.1 NA toluene 
12.7 &lt;0.5% ditert-butyloxamide B 
______________________________________ 
EXAMPLE 6 
##STR5## 
Piperidine-4-carboxaldehyde-1-benzyl carbamate (5) 
______________________________________ 
Materials: 
______________________________________ 
Isonipecotic acid chloride N-benzyl carbamate (4) 
3.38 kg (12.0 
mol) 
in toluene (MW = 281.74) 
in 5.54 kg 
DIEA (KF = 18 mg/L) 1.55 kg (15.0 mol) 
10% Pd/C (KF &lt;20 mg/g) 101 g 
thioanisole (MW = 124.21, d = 1.058) 
0.56 g 
______________________________________ 
The DIEA and thioanisole were added to the solution of (4) in toluene from 
the previous step and the catalyst was suspended in this mixture. The 
mixture was immediately placed into the 5 gal autoclave and hydrogenated 
at 20.degree. C. and 40 psi of H.sub.2. After 18 h the reaction had taken 
up 70% the theoretical amount of hydrogen and HPLC analysis of an aliquot 
that was quenched with tert-butylamine indicated that 14.2 area % of acid 
chloride 2 remained. HPLC conditions same as above. Retention time: 5=8.1 
min. 
A second charge of catalyst (101 g) and thioanisole (0.54 g) were added as 
a slurry in 1375 mL toluene to the hydrogenator. After 23 h HPLC analysis 
of an aliquot that was quenched with tert-butylamine indicated that 1.8 
area % of acid chloride 2 remained. The mixture was purged with nitrogen 
and the catalyst and precipitated DIEA.multidot.HCl were removed by 
filtration through Solka-floc.TM.. The filter cake was washed with 10 L of 
toluene. The filtrates were transferred through a 10.mu. inline filter to 
a 50 L extractor and washed with 2.times.7.2 L of 1 M aqueous HCl and 
2.times.7.2 L of water. The mixture was concentrated at 10 mbar and a 
temperature of 25-30.degree. C. until 5 L of residue remained. 
______________________________________ 
Retention time (min) 
Area % Identity 
______________________________________ 
2.1 &lt;2 carboxylic acid 3 
6.6 &lt;1 dimer 21 
8.1 &gt;95 aldehyde 5 
______________________________________ 
The assay yield of aldehyde 3 was 94% by HPLC analysis. 
EXAMPLE 7 
##STR6## 
CBZ-Spiroindoline (9) 
______________________________________ 
Materials: 
______________________________________ 
Piperidine-4-carboxaldehyde-1-benzyl 
1.71 kg (6.89 mol) 
carbamate (5) in toluene solution 
in 21.4 kg 
Phenylhydrazine 900 mL, 981 g (9.15 mol) 
Trifluoroacetic acid (TFA) 
2.20 L, 3.26 kg (28.6 mol) 
NaBH.sub.4 300 g, (7.93 mol) 
Toluene 34.4 kg 
MeCN 7.0 L 
MeOH 7.0 L 
______________________________________ 
The crude aldehyde 5 solution from the previous step was transferred 
through a 10.mu. inline filter to a 100 L reactor equipped with Teflon 
coated copper coils for cooling or heating and a mechanical stirrer. 
Toluene (34.4 kg) and MeCN (7 L) were added, and the resulting solution 
was cooled to 0.degree. C. Phenylhydrazine was added in portions and the 
temperature was maintained at -1 to 3.degree. C. while nitrogen was 
continuously bubbled through the reaction mixture. 
The phenylhydrazine was added until TLC and HPLC analysis indicated 
complete consumption of the aldehyde 5 and the appearance of a slight 
excess (&lt;5%) of phenylhydrazine. TLC conditions: Silica, E. Merck 
Kieselgel G60 F254 0.25 mm; diethyl ether/pentane (4/1); and developing 
agent 0.5% ceric sulfate, 14% ammonium molybdate in 10% aqueous sulfuric 
acid then heat; Rf: aldehyde 5=0.52, phenylhydrazone 7=0.61, 
phenylhydrazine 6=0.21. 
HPLC conditions: 25 cm Dupont Zorbax RXC8 column at 30.degree. C. with 1.0 
mL/min flow and detection at 254 nm; gradient schedule: 
______________________________________ 
Time (min) acetonitrile:water 
______________________________________ 
0 57:43 
10 65:35 
15 75:25 
18 75:25 
______________________________________ 
retention times: phenylhydrazine 6=4.5 min, toluene=7.2 min, 
phenylhydrazone 7=11.4 min. 
The reaction mixture was aged for 30 min at 0-2.degree. C., and TFA was 
added maintaining the temperature between 2 and 7.degree. C. The reaction 
mixture was warmed to 50.degree. C. over 30 min, and maintained for 17 h. 
The nitrogen sparge through the reaction mixture was stopped and a slow 
stream of nitrogen was maintained over the reaction mixture. During the 
first hour at 5.degree. C. the color gradually darkened to a deep green, 
and a relatively small amount of a white crystalline precipitate (ammonium 
trifluoroacetate) formed. After 17 h HPLC analysis (same conditions as 
above) indicated that the reaction mixture contained 91.6 area % 
indolenine 8 and 1.5% of unreacted phenylhydrazone remained. Aging the 
mixture for longer periods of time did not increase the assay yield of 
indolenine 8. 
The reaction mixture was cooled to 12.degree. C., and 7.0 L of MeOH was 
added. NaBH.sub.4 was added in small (&lt;20 g) portions maintaining the 
temperature below 15.degree. C. The addition took 30 min. Moderate 
hydrogen evolution was observed during the addition, but it was easily 
controlled and there was virtually no frothing. Near the end of the 
addition the color rapidly changed from green to brown and then bright 
orange. A small amount (&lt;200 mL) of a heavier phase had separated 
(presumably aqueous salts). HPLC analysis (conditions as before) indicated 
that all of the indolenine 8 had been consumed (90.4 area % CBZ-indoline 
9); retention times: indolenine 8=7.5 min, indoline 9=8.2 min. TLC: ethyl 
ether as solvent, ceric sulfate-ammonium molybdate stain or 1% 
anisaldehyde stain; retention factors: indolenine 8=0.18, CBZ-indoline 
9=0.33. 
The color change from green to orange corresponds very closely to reaction 
end point. The quantity of NaBH.sub.4 required to complete the reaction is 
heavily dependent on the temperature and rate of addition of NaBH.sub.4, 
but the yield and quality of the product is virtually unaffected provided 
that the reaction is complete. The reaction mixture was cooled to 
5.degree. C. over a period of 30 min. Then 8 L of 3% aqueous NH.sub.4 OH 
(8 L) were added to bring the pH of the aqueous phase to 7.4, the mixture 
was agitated, and allowed to settle. The temperature rose to 15.degree. C. 
The cloudy yellow lower aqueous phase was separated. The organic phase was 
washed with 4 L of 3% aqueous NH.sub.4 OH, 2.times.4 L of water, and 
2.times.4 L of brine. The weight of the organic phase after the washings 
was 53.5 kg, and the assay yield was 94%. 
The washed toluene solution was combined with the washed organic phases of 
two other similarly processed reactions. The total aldehyde used in the 
three reactions was 5.06 kg, (20.5 mol). The total weight of CBZ-indoline 
9 assayed in the combined organic phases was 5.91 kg, (18.3 mol, 90% assay 
yield). The combined organic phases were dried with 5 kg of sodium 
sulfate, treated with 250 g of Darco G60 carbon for 30 min, and filtered 
through Solka-floc.TM.. The filtrates were vacuum concentrated at 10 mbar 
at &lt;25.degree. C. until the residue was near dryness. The solvent switch 
was completed by slowly bleeding in 30 L of I and reconcentrating to 14 
L at 200 mbar at 50-60.degree. C. The mixture was heated to reflux in 
order to obtain a clear homogeneous deep orange solution. .sup.1 H NMR 
analysis indicated that the solution contained ca. 6 mol % of residual 
toluene after solvent switch. 
The solution was cooled to 68.degree. C. and seeded with 4 g of crystalline 
CBZ-indoline 9. The solution was allowed to gradually cool to 26.degree. 
C. over 6 h and aged for 9 h at 20-26.degree. C. The slurry was cooled to 
2.degree. C. over 1 h and aged at 2.degree. C. for 1 h. The product was 
isolated by filtration, and the filter cake was washed with 2.times.2 L of 
5.degree. C. I and 2.times.2 L of 5.degree. C. MTBE. The product was 
dried in the vacuum oven at 30.degree. C. under a nitrogen bleed to give 
4.37 kg (74%) of the title compound 9 as a light tan crystalline powder. 
HPLC analysis of the product indicated 99.5 area % purity. The mother 
liquor (11 L) and the washes contained 1.15 kg (19%) of additional product 
9 and ca 3% of Cbz-isonipecotic acid phenylhydrazide (retention time=4.8 
min). 
EXAMPLE 8 
##STR7## 
CBZ-Spiroindoline-methanesulfonamide (1) 
______________________________________ 
Materials: 
______________________________________ 
CBZ-Spiroindoline (9) 
1.69 kg (5.23 mol) 
Methanesulfonyl chloride 
599 g (5.23 mol) 
Et.sub.3 N (KF = 151) 
635 g (6.27 mol) 
THF (KF = 41) 12 L 
______________________________________ 
A 22 L flask was charged with the solid CBZ-spiroindoline 9 and then 11.5 L 
of THF and the Et.sub.3 N were transferred into the flask through a 10.mu. 
inline filter. The resulting homogenous solution was cooled to 0.degree. 
C. A 1 L dropping funnel was charged with the methanesulfonyl chloride and 
500 mL of THF. The solution of the MsCl in THF was added to the reaction 
mixture maintaining the temperature between 0 and 4.degree. C. The 
addition took 5 h and was exothermic. A white precipitate, presumably 
triethylammonium hydrochloride formed during the addition. HPLC analysis 
indicated that the reaction was complete at the end of the addition (9 was 
undetectable. 
HPLC conditions: 25 cm Dupont Zorbax RXC8 column with 1.5 mL/min flow and 
detection at 254 nm. Gradient Schedule: 
______________________________________ 
Time (min) 0.1% aq. H.sub.3 PO.sub.4 :MeCN 
______________________________________ 
0 70:30 
3 70:30 
12 20:80 
25 20:80 
______________________________________ 
Retention times: 9=7.6 min, 1=13.6 min. 
After the addition was complete the reaction mixture was warmed to 
18.degree. C. and aged for 16 h. There was no change in the appearance of 
the reaction mixture, and HPLC profile between the end of the addition and 
after the 16 h age. The reaction mixture was slowly transferred over 1 h 
into a vigorously stirred solution of 30 L of water and 200 mL of 37% 
aqueous HCl in a 50 L flask. The temperature in the 50 L flask rose from 
22 to 28.degree. C. The product separated as a pale tan gummy solid which 
changed to a granular solid. The aqueous suspension was cooled to 
22.degree. C. and aged for 1 h. The suspension was filtered, and the 
filter cake was washed with 2.times.4 L of MeOH/water (50/50). HPLC 
analysis indicated that &lt;0.1% of the CBZ-Spiroindoline-methanesulfonamide1 
was in the mother liquors. 
The filter cake was washed with 4 L of MeOH/water (50/150) to which 50 mL 
of 28% aqueous NH.sub.4 OH had been added. The filter cake was washed with 
2.times.4 L of MeOH/water (50/50), and the solid was dried in the vacuum 
oven at 50.degree. C. under a nitrogen bleed to give 2.03 kg (97%) of the 
title product 1 as an off-white powder. HPLC analysis of the solids 
indicated 93.7 area % 1. 
EXAMPLE 9 
##STR8## 
Optional Procedure for Isolation of Intermediate CBZ-Spiroindolenine (8) 
______________________________________ 
Materials: 
______________________________________ 
Piperidine-4-carboxaldehyde-1-benzyl 
12.37 g (0.050 mol) 
carbamate (5) 
Phenylhydrazine 5.41 g (0.050 mol) 
Trifluoroacetic acid (TFA) 
11.56 mL, 17.10 g 
(0.150 mol) 
Methylene chloride 500 mL 
______________________________________ 
The CBZ-aldehyde 5 was dissolved in dichloromethane in a 1 L flask equipped 
with Teflon coated magnetic stirring bar. The resulting solution was 
cooled to 0.degree. C. Phenylhydrazine was added via a weighed syringe 
over 5 min and the temperature was maintained at -1 to 3.degree. C. while 
nitrogen was continuously bubbled through the reaction mixture. TLC and 
HPLC analysis indicated complete consumption of the CBZ-aldehyde 5 and the 
appearance of a slight excess (&lt;2%) of phenylhydrazine. TLC conditions: 
Silica, E. Merck Kieselgel G60 F254 0.25 mm; diethyl ether/pentane (4/1); 
and developing agent 0.5% ceric sulfate, 14% ammonium molybdate in 10% 
aqueous sulfuric acid then heat; Rf: aldehyde 5=0.52, phenylhydrazone 
7=0.61, phenylhydrazine 6=0.21. HPLC conditions: 25 cm Dupont Zorbax RXC8 
column at 30.degree. C. with 1.0 mL/min flow and detection at 254 nm; 
gradient schedule: 
______________________________________ 
Time (min) acetonitrile:water 
______________________________________ 
0 57:43 
10 65:35 
15 75:25 
18 75:25 
______________________________________ 
retention times: phenylhydrazine 6=4.5 min, toluene=7.2 min. 
phenylhydrazone 7=11.4 min. 
The reaction mixture was aged for 10 min at 0-2.degree. C., and TFA was 
added by syringe maintaining the temperature between 2 and 7.degree. C. 
The reaction mixture was warmed to 35.degree. C. over 30 min, and 
maintained for 17 h. The nitrogen sparge through the reaction mixture was 
stopped and a slow stream of nitrogen was maintained over the reaction 
mixture. During the first hour at 35.degree. C. the color gradually 
darkened to a rosy pink then to a deep green, and a relatively small 
amount of a white crystalline precipitate (ammonium trifluoroacetate) 
formed. After aging for 17 h HPLC analysis (same conditions as above) 
indicated that the reaction mixture contained 93 area % indolenine 8 and 
&lt;0.5% of unreacted phenylhydrazone remained. Aging the mixture for longer 
periods of time did not increase the assay yield of indolenine 8. The 
reaction mixture was cooled to 10.degree. C., and a mixture containing 60 
mL 28-30% ammonium hydroxide, 90 mL water and 150 g crushed ice was added 
with good stirring. The color of the mixture changed to a salmon color. 
The organic phase was separated and washed twice with 400 mL water then 
100 mL saturated aqueous NaCl. The organic phase was dried over magnesium 
sulfate and filtered through a plug of 5 g of silica. The filtrate was 
evaporated to give 15.84 g (99%) of indolenine 8 as a pale orange oil. 
EXAMPLE 10 
##STR9## 
Procedure for the Preparation of CBZ-Spiroindoline-methanesulfonamide (1) 
without Isolation of Intermediate CBZ-Spiroindoline (9) 
Step 1: CBZ-Spiroindoline (9) 
______________________________________ 
Materials: 
______________________________________ 
Piperidine-4-carboxaldehyde-1-benzyl 
49.5 g (0.20 mol) 
carbamate (5) 
Phenylhydrazine (Aldrich) 
23.7 g (0.22 mol) 
Trifluoroacetic acid (TFA) 
75.4 g (0.66 mol) 
Toluene (KF &lt;250 mg/L) 
654 mL 
MeCN (KF &lt;250 mg/L) 13.3 mL 
NaBH.sub.4 11.3 g, (0.30 mol) 
Toluene 20 mL 
MeOH 50 mL 
______________________________________ 
A 2% (by volume) solution of MeCN in toluene was made up using 654 mL of 
toluene and 13.3 mL of MeCN. In a 2 L 3 neck flask equipped with a 
mechanical stirrer 617 ml of the above solution were degassed by passing a 
fine stream of nitrogen through the solution for 5 min. Phenylhydrazine 
and TFA were added to the mixture while still degassing. 
The CBZ-aldehyde 5 was dissolved in the rest of the solution prepared above 
(50 mL) and degassed by bubbling nitrogen through the solution while in 
the addition funnel. The solution in the flask was heated to 35.degree. 
C., and the aldehyde solution was slowly added to the phenylhydrazine-TFA 
over 2 h. The mixture was aged at 35.degree. C. for 16 h. 
HPLC conditions: 25 cm Dupont Zorbax RXC8 column at 50.degree. C. with 1 
mL/min flow and detection at 220 nm; isocratic 55% MeCN, 45% of 0.1% 
aqueous H.sub.3 PO.sub.4. Typical HPLC profile after 16 h age: 
______________________________________ 
Retention time (min) 
Area % Identity 
______________________________________ 
1.6 0.1-0.5 phenylhydrazine 6 
4.1 &lt;0.1 dimer 21 
4.7 &lt;0.1 aldehyde 5 
5.0 NA spiroindoline 9 
6.3 NA toluene 
6.9 97 spiroindolenine 8 
10.3 &lt;0.2 phenylhydrazone 7 
2-3 tot. other impurities &lt;0.2% ea. 
______________________________________ 
The mixture was cooled to -10.degree. C. and MeOH was added. A suspension 
of sodium borohydride in 20 mL toluene was added in small portions (1 mL) 
over 30 min taking care that the temperature did not exceed -2.degree. C. 
______________________________________ 
Area % Identity 
______________________________________ 
0.1-1 phenylhydrazine 6 
85-90 CBZ-spiroindoline 9 
&lt;0.1 CBZ-spiroindolenine 8 
10-15 tot. other impurities (&lt;3% ea.) 
______________________________________ 
The temperature was raised to 10.degree. C. over 1 h, and 6% aqueous 
ammonia (200 mL) was added. The mixture was agitated for 10 min, allowed 
to settle for another 10 min, and the lower aqueous phase was drawn off. 
Acetonitrile (20 mL) and MeOH (20 mL) were added to the organic phase and 
it was washed with 150 mL of 15% brine. The organic phase was found to 
contain a 92% assay yield of CBZ-spiroindoline 9. 
Step 2: CBZ-Spiroindoline-methanesulfonamide (1) 
______________________________________ 
Materials: 
______________________________________ 
CBZ-Spiroindoline (9) (MW = 322.51) 
(0.184 mol) 
Methanesulfonyl chloride 
21.1 g (0.18.4 mol) 
DIEA (KF = 150 mg/L) 29.7 g, 40.1 mL 
(0.230 mol) 
THF (KF = 41 mg/L) 150 mL 
______________________________________ 
The crude solution of CBZ-spiroindoline 9 solution from Step 1 above was 
concentrated in a 1 L 3 neck flask (60-70.degree. C., 150-200 Torr) until 
250 g of residue remained. The THF and DIEA were added, and the resulting 
homogenous solution was cooled to 0.degree. C. A 125 mL dropping funnel 
was charged with the methanesulfonyl chloride and 50 mL of THF. The 
solution of MsCl in THF was added over 2 h to the reaction mixture 
maintaining the temperature between 0 and 4.degree. C. and the mixture was 
aged for 2 h at 5-8.degree. C. The addition was slightly exothermic. A 
white precipitate, presumably DIEA-hydrochloride, formed during the 
addition. HPLC conditions were the same as above. HPLC analysis indicated 
that the reaction was complete 1 h after the end of the addition (9 was 
undetectable) and the assay yield was 94% from 9. Retention time: 1=7.8 
min. Typical HPLC profile of reaction mixture after 2 h age: 
______________________________________ 
Area % Identity 
______________________________________ 
&lt;0.1 CBZ-spiroindoline 9 
90-92 CBZ-sulfonamide 1 
8-10 tot. other impurities (&lt;2% ea.) 
______________________________________ 
The mixture was warmed to 20.degree. C., and 200 mL of 1M aqueous HCl was 
added. The mixture was warmed to 50.degree. C., and the aqueous phase was 
separated. The organic phase was washed sequentialy with 100 mL water, 100 
mL 5% aqueous sodium bicarbonate, and 100 mL water. The organic phase was 
transferred to a 1 L 3 neck flask equipped for mechanical stirring and 
distillation. The mixture (ca 400 mL) was distilled at atmospheric 
pressure until 150 mL of distillate had been collected. The head 
temperature reached 107.degree. C.; the pot temperature was 110.degree. C. 
The distillation was continued with continuous addition of n-propanol at 
such a rate as to maintain a constant volume (ca 350 mL) in the pot. The 
distillation was stopped when a total of 525 mL of n-PrOH had been added 
and a total of 800 mL of distillate had been collected. 
The temperature of both the head and pot rose from 94.degree. C. to 
98.degree. C. during the solvent switch. Toluene and n-PrOH form an 
azeotrope boiling at 97.2.degree. C. composed of 47.5% toluene and 52.5% 
n-PrOH. The mixture was allowed to cool gradually to 20.degree. C. over 3 
h and aged for 12 h. The mother liquor was found to contain 2% toluene and 
4 mg/mL of sulfonamide. The solubility of the sulfonamide in various 
mixtures of toluene and n-PrOH has been determined by HPLC assay: 
______________________________________ 
% toluene in n-PrOH 
solubility of 1 in mg/mL 
______________________________________ 
0 2.36 
5 3.02 
10 4.23 
20 7.51 
25 10.3 
______________________________________ 
The crystalline slurry was filtered and washed with 3.times.100 mL of 
n-PrOH. The product was dried in a vacuum oven at 50.degree. C. with a 
nitrogen bleed for 16 h to furnish 65.5 g (82% from aldehyde 5) of 6 as a 
tan solid with 93.5 wt % purity. 
Typical HPLC profile of solid: 
______________________________________ 
Area % Identity 
______________________________________ 
&lt;0.1 CBZ-spiroindoline 9 
&gt;99 CBZ-sulfonamide 1 
&lt;1 tot. other impurities (&lt;0.2% ea.) 
______________________________________ 
For additional purification, a 40.0 g sample of the n-PrOH crystallized 
sulfonamide was dissolved in 134 mL of EtOAc at 60.degree. C. and treated 
with 8.0 g of Darco G-60 carbon for 1 h at 60.degree. C. After the 
addition of 2.0 g Solkafloc.TM., the slurry was filtered through a pad of 
4.0 g Solkafloc.TM., and the pad was washed with 90 mL of EtOAc at 
60.degree. C. Prior to the addition of the carbon the solution was a brown 
color. The filtration proceeded well without plugging to give a golden 
yellow filtrate. The filtrate was distilled at atmospheric pressure in a 
500 mL flask (pot temperature 80-85.degree. C.) until 100 g (100 mL) of 
residue remained. This solution was allowed to cool to 35.degree. C. over 
3 h. Over a 1 h period, 116 mL of cyclohexane was added with good 
agitation at 35.degree. C. The mixture was cooled to 20.degree. C. over 1 
h and aged at 20.degree. C. for 12 h. At 35.degree. C. much of the 
sulfonamide has crystallized out and the mixture was thick. Addition of 
cyclohexane at 20.degree. C. makes agitation difficult. After the aging 
period, the supernatant was found to contain 2.5 mg 1/g. The crystalline 
slurry was filtered and the cake was washed with 77 mL of 2:1 
cyclohexane-EtOAc and 2.times.77 mL of cyclohexane. The product was dried 
in a vacuum oven at 50.degree. C. with a nitrogen bleed for 16 h to 
furnish 34.2 g of 1 (MW=400.3) as a white crystalline solid (85% recovery 
from crude 1, 70% from 5 with &gt;99.9 wt % purity). 
EXAMPLE 11 
##STR10## 
HCl Salt of Spiroindoline-methanesulfonamide (1a) 
______________________________________ 
Materials: 
______________________________________ 
CBZ-spiroindoline-methanesulfonamide (1) 
941 g (2.35 mol) 
Pearlman's catalyst 20% Pd(OH).sub.2 /C 
188 g 
THF 8 L 
MeOH 7 L 
______________________________________ 
The catalyst was suspended in 7 L of MeOH and transferred into the 5 gal 
autoclave followed by the solution of 1 in 8 L of THF. 
The mixture was hydrogenolyzed at 25.degree. C. at 80 psi of H.sub.2. After 
2.5 h the temperature was raised to 35.degree. C. over 30 min. 
HPLC analysis indicated complete consumption of 
Cbz-spiroindoline-methanesulfonamide. HPLC conditions: 25 cm Dupont Zorbax 
RXC8 column with 1.5 mL/min flow and detection at 254 nm. Gradient 
Schedule: 
______________________________________ 
Time (min) 0.1% aq. H.sub.3 PO.sub.4 :MeCN 
______________________________________ 
0 70:30 
3 70:30 
12 20:80 
25 20:80 
______________________________________ 
retention times: Spiroindoline = 7.6 min, 
Cbzspiroindoline-methanesulfonamide = 13.6 min. 
The mixture was purged with nitrogen and the catalyst was removed by 
filtration through Solka-floc.TM. while still warm. The catalyst was 
washed with 4 L of THF and 2 L of MeOH. The pale yellow filtrates were 
concentrated to a thick oil at 10 mbar and &lt;25.degree. C. The solvent 
switch was completed by slowly bleeding in 15 L of EtOAc and 
reconcentrating to dryness. The residue solidified to a hard off-white 
mass. MeOH (1.5 L) was added and the mixture was heated to 70.degree. C. 
to give a homogenous solution. While the solution was at 70.degree. C., 
10.5 L of EtOAc at 20.degree. C. was added. The temperature fell to 
40.degree. C., and the mixture remained homogenous. 
Subsequent experiments suggested that it is more convenient to solvent 
switch the MeOH-THF filtrates to. MeOH, concentrate to the desired volume, 
and then add the EtOAc. This avoids the solidification of the residue upon 
concentration of the EtOAc solution. 
Hydrogen chloride diluted with about an equal volume of nitrogen was passed 
into the solution. The temperature rose to 60.degree. C. over the course 
of 15 min, and a white precipitate of the hydrochloride salt formed. 
Diluting the HCl with nitrogen only avoids the reaction mixture sucking 
back and may not be necessary. 
The mixture was cooled in an ice bath, and the hydrogen chloride addition 
was continued for 1 h. The temperature gradually fell to 20.degree. C. The 
suspension was aged for 2 h while the temperature was lowered to 10IC. The 
crystalline product was isolated by filtration, and the filter cake was 
washed with 3 L of EtOAc. It was dried in the vacuum oven at 35.degree. C. 
to give 1.18 kg (86%) of the title product 1 a as an off-white crystalline 
solid of &gt;99.5 area % purity by HPLC analysis. HPLC conditions: 25 cm 
Dupont Zorbax RXC8 column with 1.5 mL/min flow and detection at 230 nm; 
isocratic 35% MeCN, 65% of 0.1% aqueous ammonium acetate. Retention time: 
1a=5.4 min. 
EXAMPLE 12 
##STR11## 
Spiroindoline-methanesulfonamide (Free base form) (1b) 
A 250 mL aliquot of the filtrate from the Cbz-hydrogenolysis containing 
4.67 g of 1b (free base) was concentrated to ca 10 mL. The residue was 
dissolved in 20 mL of EtOAc and the solution was reconcentrated to ca 10 
mL. This was repeated once more, and 10 mL of EtOAc was added to the 
residue. A crystalline precipitate began to form. MTBE (20 mL) was added 
in one portion. Additional crystalline solid precipitated, but the 
supernatent still contained a substantial quantity of dissolved product 
which did not precipitate on standing. Hexanes (70 mL) were added dropwise 
over 2 h to the mixture with vigorous stirring. The slow addition of the 
hexanes is neccessary to avoid the oiling out of the amine. The agitated 
mixture was aged for 1 h and filtered. The filter cake was washed with 20 
mL of 1:1 MTBE-hexanes and then with 20 mL of hexanes. The product was 
dried under a stream of nitrogen to give 3.86 g (82%) of the free amine of 
1b as an off white crystalline solid of &gt;99.5 area % purity. HPLC 
conditions: 25 cm Dupont Zorbax RXC8 column with 1.5 mL/min flow and 
detection at 230 nm; isocratic 35% MeCN, 65% of 0.1% aqueous ammonium 
acetate. Retention time: 1b=5.4 min. 
EXAMPLE 13A 
##STR12## 
Spiroindoline-methanesulfonamide (Free base form) (1b) 
______________________________________ 
Materials: 
______________________________________ 
CBZ-Spiroindoline-sulfonamide (1) 
833.5 g (2.08 mol) 
Pd(OH).sub.2 /C (20% weight of Pd(OH).sub.2) 
124.5 (15%) 
THF 6.5 L 
MeOH 19.5 L 
NH.sub.4 OH (conc) 60 mL 
______________________________________ 
The hydrogenation was run three (3) times due to equipment limitations; 
this procedure refers to a single run. The CBZ spiroindoline sulfonamide1 
was dissolved in THF (6.5 L, KF=53 .mu.g/.mu.L) and then MeOH (KF=18 
.mu.g/mL, 4 L) was added followed by addition of the catalyst and the 
slurry was transferred to a 5 gal autoclave. The remainder of the MeOH 
(2.5 L) was used for rinsing. The mixture was heated to 40.degree. C. at 
50 psi for 24 hours. The catalyst loading and reaction time are a function 
of the purity of starting material 1. This material was unique requiring 
.gtoreq.15% catalyst and long reaction time. Purer batches of 
spiroindoline required only 5% of catalyst and 4-6 hrs reaction time. 
Upon completion (&lt;0.1 A % 1 by LC) the mixture was filtered thru Solka 
Floc.TM. and the carbon cake washed with MeOH (13 L) containing NH.sub.4 
OH (0.5%, 60 mL). The combined filtrates (assay shows 1587 g of 
spiroindoline amine 1b) were concentrated in vacuo and the resulting 
solids were partitioned between 40 L (of toluene:THF (3:1) and 0.5N NaOH 
(18 L). Although the layers separated easily a heavy precipitate could be 
seen in the aqueous layer. The aqueous suspension was thus extracted with 
CH.sub.2 Cl.sub.2 (15 L). The aqueous and organic layer separated slowly. 
Prior to CH.sub.2 Cl.sub.2 addition THF was added to the aqueous layer 
along with enough NaCl to saturate the layer. However dissolution of the 
product was not achieved which necessitated the use of CH.sub.2 Cl.sub.2. 
The combined toluene, THF and CH.sub.2 Cl.sub.2 layers were combined and 
concentrated in the batch concentrator. The residue was flushed with 7 L 
of CH.sub.3 CN. Finally 10 L of CH.sub.3 CN were added and the solution 
stood overnight under N.sub.2 atmosphere. 
EXAMPLE 13B 
##STR13## 
Spiroindoline-methanesulfonamide (Free base form) (1b) 
______________________________________ 
Materials: 
______________________________________ 
CBZ-Spiroindoline-sulfonamide (1) 
3 kg (7.49 mol) 
Darco G-60 600 g 
Ethyl Acetate 36 L 
Absolute Ethanol 189 L 
10% Pd/C 450 g 
Ammonia Solution 500 ml 
Solka Floc .TM. 2.5 kg 
Isopropyl Acetate 65 L 
______________________________________ 
A mixture of CBZ-spiroindoline (1) (1 kg) and Darco G-60 (200 g) in ethyl 
acetate (9 L) was stirred and heated at 60-65.degree. C. under a nitrogen 
atmosphere for 8 hours. The Darco was removed by filtration at 
60-65.degree. C., the solid washed with hot ethyl acetate (3 L) and the 
filtrate and washings combined. LC wt/wt assay confirmed negligible loss 
to the Darco. The ethyl acetate solution was evaporated to dryness in 
vacuo using a 20 L Buchi apparatus and then flushed with absolute ethanol 
(2.times.5 L). This material was then slurried in absolute ethanol (8 L) 
warmed to 65-70.degree. C. and placed in the 20 L autoclave. The batch was 
rinsed in with absolute ethanol (1 L). A slurry of 10% Palladium on 
charcoal (75 g, 7.5% by weight) in absolute ethanol (750 ml) was then 
added to the autoclave and rinsed in with a further portion of absolute 
ethanol (250 ml). 
The batch was hydrogenated at 65.degree. C. with vigorous stirring under 40 
psi hydrogen pressure for 3 hours, a second portion of 10% palladium on 
charcoal (75 g) was added, the batch was hydrogenated for a further 2 
hours and then sealed overnight. The batch was transferred (still hot, 
60-65.degree. C.) to a 20 L Buchi apparatus and degassed in vacuo to 
remove formic acid by "feeding and bleeding" absolute ethanol (18 L 
total). 
This procedure was repeated twice more and the three batches were combined 
in a 10 gallon glass-lined vessel and the combined batch was degassed 
again by the addition and distillation (in vacuo) of absolute ethanol 
(2.times.10 L). Solka floc.TM. (0.5 kg) was added to the batch and rinsed 
in with ethanol (10 L). An Estrella filter was loaded with Solkafloc.TM. 
(2 kg) as a slurry in ethanol (20 L). The resulting mixture was warmed to 
60-65.degree. C. and then transferred at this temperature via heated 
filter using pump to two tared stainless-steel bins. The initial vessel, 
the filter, the pump and the lines were rinsed with a hot (60-65.degree. 
C.) mixture of aqueous ammonia (500 ml) in absolute ethanol (25 L). The 
filtrate and washings were combined in the two stainless-steel bins. 
The batch was then transferred to a vessel using an in-line filter 
containing a 10 micron cartridge, and then concentrated in vacuo to low 
bulk (.about.15 L). The ethanol was replaced by isopropyl acetate by the 
"feeding and bleeding" of 3x batch volumes of isopropyl acetate (45 L 
total), while maintaining a batch volume of .about.15 L. The solvent 
switch, when complete, contained &lt;1% residual ethanol by GC. The batch was 
then diluted to .about.33 L by the addition of isopropyl acetate (20 L), 
and this solution of spiroindoline-amine 1b (1.855 kg by LC analysis) in 
isopropyl acetate was used for the next stage of the process. 
EXAMPLE 14A 
##STR14## 
Boc-O-Benzylserine Spiroindoline (11) 
______________________________________ 
Materials: 
______________________________________ 
Spiroindoline-amine (1b) 
1587 g (5.966 moles) 
Amino acid (10) 1938 g (6.563 moles) 
##STR15## 
DCC 1334.5 g (6.563 moles) 
HOBT 884 g (6.563 moles) 
CH.sub.3 CN 25 L 
0.5N NaOH 18 L 
0.5N HCl 18 L 
NaHCO.sub.3 sat. 18 L 
iPrOAc 28 L 
______________________________________ 
The spiroindoline-amine1b in CH.sub.3 CN or iPrOAc:H.sub.2 O (25 L) at 
ambient temperature under N.sub.2 was treated in sequence with HOBT (884 
g; 1.1 eq) as a solid, DCC (1334.5 g, 1.1 eq) as the melt (heating in hot 
water at 60.degree. C. for ca. 1 hr) and finally the amino acid 10 (1938 
g) as the solid. The mixture was stirred for 3 hr upon which time heavy 
precipitation of DCU occurred and LC analysis showed ca. 0.5 A % of amine 
1b remaining. IPAc (9 L) was added, the slurry was filtered through Solka 
Floc.TM. and the cake was washed with IPAc (19 L). The combined organic 
solution was washed in sequence with 0.5N NaOH (18 L), 0.5N HCl (18 L) and 
saturated NaHCO.sub.3 (18 L). A final water wash at this point resulted in 
an emulsion and was thus eliminated. 
The organic layer was concentrated in vacuo and the residue was dissolved 
in MeOH or EtOH (10 L final volume). Assay yield 3026 gr (89%). 
The use of alternative peptide coupling agents such as carbonyldiimidazole 
or formation of mixed anhydrides, such as sec-butyl carbonate, gave 
inferior yields of 11 and/or 14 with a high degree of epimerization in the 
case of the former compound. Other peptide coupling reagents were 
prohibitively expensive. 
EXAMPLE 14B 
##STR16## 
Boc-O-Benzylserine Spiroindoline (11) 
______________________________________ 
Materials: 
______________________________________ 
Spiroindoline-amine (1b) 
1.855 kg (6.96 mol) 
Isopropyl acetate 29 L 
Dicyclohexylcarbodiimide (DCC) 
1.58 kg (7.65 mol) 
1-Hydroxybenzotriazole (HOBt) 
1.03 kg (7.62 mol) 
N-Boc-O-benzyl-D-Serine 
2.26 kg (7.65 mol) 
1 M Aqueous sodium hydroxide 
26 L 
0.5 M Aqueous hydrochloric acid 
26 L 
Satd. Aqueous sodium hydrogen carbonate 
26 L 
Absolute Ethanol 50 L 
______________________________________ 
Water (20 L) was added to a stirred solution of the spiroindoline-amine1b 
(1.855 kg) in isopropyl acetate (33 L) in a reaction vessel. The following 
chemicals were then added sequentially at room temperature under a 
nitrogen atmosphere: DCC (1.58 kg, 1.1 equivs.), HOBt (1.03 kg, 1.1. 
equivs.) and finally N-Boc-O-benzyl-D-Serine (2.26 kg, 1.1 equivs.). The 
reagents were rinsed in with isopropyl acetate (7 L). The batch was 
stirred at room temperature under nitrogen atmosphere for 5 hours when LC 
showed the ratio of product/starting material to be 99.4/0.6. The mixture 
was then filtered through an Estrella filter using cloth and cardboard 
only and utilizing a pump into another vessel. The sending vessel was 
rinsed with isopropyl acetate (22 L) and this was used to rinse the 
filter, the pump and the lines into the receiving vessel. The 2-phase 
mixture in the vessel was stirred for 10 minutes and then allowed to 
settle for 15 minutes. The lower aqueous layer was separated off and the 
organic solution was left to stand at room temperature overnight. 
The next day, the organic solution was washed with 1M aqueous sodium 
hydroxide solution (26 L) then 0.5M aqueous hydrochloric acid (26 L) and 
finally saturated aqueous sodium hydrogen carbonate (26 L). LC analysis 
gave an assay yield of 3.787 kg, 93% overall yield from 7.49 moles (3 kg) 
of starting CBZ-spiroindoline (1). The batch was concentrated in vacuo 
(internal temperature=13-15.degree. C. jacket temperature=40.degree. C., 
Vacuum=29") to low bulk (.about.15 L) and solvent switched to ethanol by 
"feeding and bleeding" ethanol (50 L) whilst maintaining the volume at 
.about.15 L. GC showed &lt;1% isopropyl acetate remaining. This solution was 
used for the next stage of the process. 
EXAMPLE 15A 
##STR17## 
O-Benzylserine Spiroindoline (free base form) (12) 
______________________________________ 
Materials: 
______________________________________ 
Boc-O-Benzylserine Spiroindoline (11) 
3026 g (5.57 moles) 
Methane sulfonic acid (MsOH) 
1.16 L (17.9 moles) 
MeOH 10 L 
iPrOAc 24 L 
0.5 N NaOH 35 L 
______________________________________ 
The Boc-O-benzylserine spiroindoline 11 in 10 L of MeOH (or EtOH) was 
treated with neat MsOH (1.16 L) added over ca. 30-40 min, (initial 
temperature 16.degree. C., final temperature 28.degree. C.). The dark red 
solution was aged overnight under N.sub.2. The mixture was then pumped 
into a 100 L extractor containing 24 L iPrOAc and 35 L 0.5 N NaOH. The pH 
of the aqueous layer was 7. NaOH (6M) was added until pH .gtoreq.10.5. As 
the pH increased the color changed from red to yellow. The layers were 
separated and the organic layer (24 L) was shown by NMR to contain 13 mole 
% of MeOH in iPrOAc [5 volume %]. LC assay 2.48 kg. 
EXAMPLE 15B 
##STR18## 
O-Benzylserine Spiroindoline (free base form) (12) 
______________________________________ 
Materials: 
______________________________________ 
Boc-O-Benzylserine Spiroindoline (11) 
3.787 kg (6.96 mol) 
Methanesulphonic acid 2.006 kg (20.87 mol) 
Isopropyl acetate 38 L 
1 M Aqueous sodium hydroxide 
16 L 
50% Aqueous sodium hydroxide 
1.6 L 
______________________________________ 
Methanesulphonic acid (2.006 kg, 1.355 L, .about.3 equivs.) was added to 
the stirred solution of Boc-O-benzylserine spiroindoline (11) (3.787 kg) 
in ethanol (total volume -15 L) in a reaction vessel. The batch was warmed 
to 35-40.degree. C. After 7 hours, LC showed the absence of starting 
material and the reaction was allowed to cool to room temperature 
overnight. The next day, water (44 L) was added to the batch with 
stirring. The batch was cooled to .about.5.degree., stirred for 30 minutes 
and then filtered through an in-line filter (loaded with a 10.mu. 
cartridge) into a bin. The batch was then sucked back into the vessel. A 
water rinse (10 L) was used to rinse the vessel and lines into the bin and 
this was used to then rinse back into the vessel. Isopropyl acetate (38 L) 
was added followed by a 1M aqueous sodium hydroxide (16 L). The batch was 
cooled to 10-15.degree. C., the pH of the lower aqueous layer was 
confirmed as .about.7 and 50% aqueous sodium hydroxide solution was added 
(1.6 L) (pH&gt;10). The batch was stirred at 10-15.degree. C. for 25 minutes 
and then allowed to settle for 10-15 minutes. The lower aqueous layer was 
separated (78.1 kg). LC assay indicated 28.4 g of 12 (0.85% of theory) 
contained in the aqueous liquors. Volume of the organic solution=51 L. LC 
assay indicated 3.057 kg, 92% overall yield from 3 kg, 7.49 moles of 
CBZ-spiroindoline sulfonamide (1). This solution was used for the next 
stage. 
EXAMPLE 16A 
##STR19## 
Boc-Aminoisobutyryl O-Benzylserine Spiroindoline (14) 
______________________________________ 
Materials: 
______________________________________ 
Spiroindoline amine (12) 
2481 g (5.57 moles) 
amino acid peptide (13) 
1247.1 g (6.16 moles) 
##STR20## 
DCC 1266.7 g (6.16 moles) 
HOBT 827 g (6.16 moles) 
IPAc 52 L 
H.sub.2 O 37 L 
0.5N NaOH 36 L 
0.5N HCl 36 L 
Sat. NaHCO.sub.3 36 L 
______________________________________ 
The solution of the amine 12 in IPAc was diluted to a total volume of 39 L 
with IPAc and 37 L of H.sub.2 O was added. The biphasic mixture was then 
treated in sequence with HOBT (827 g) as a solid, DCC (1266.7 g) as a 
melt, and amino acid 13 at ambient temperature under nitrogen. The 
reaction mixture was stirred for 2 h upon which time LC analysis indicated 
dissappearance of the starting material 12 (&lt;0.3 A %). The mixture was 
filtered through Solka Floc.TM. and the solids were washed with 13 L of 
IPAc. The material may be stored at this point as a biphasic mixture 
overnight. 
The mixture was transferred to a 100 L extractor, the aqueous layer was 
separated and the organic layer was washed successively with 36 L of 0.5N 
NaOH, 0.5N HCl and saturated NaHCO.sub.3. Assay yield 3160 g (81% from 
spiroindoline .+-.5% for volume measurement error). The solution was 
concentrated to a small volume and was flushed with ethanol (2.times.4 L). 
If desired, the inermediate compound 14 may be isolated by adding water to 
crystalize it out. 
The use of alternative peptide coupling agents such as carbonyldumidazole 
or formation of mixed anhydrides, such as sec-butyl carbonate, gave 
inferior yields of 14 with a high degree of epimerization. Other peptide 
coupling reagents were prohibitively expensive. 
EXAMPLE 16B 
##STR21## 
Boc-Aminoisobutyryl O-Benzylserine Spiroindoline (14) 
______________________________________ 
Materials: 
______________________________________ 
Spiroindoline amine (12) 
3.057 kg (6.89 mol) 
Dicyclohexylcarbodiimide (DCC) 
1.56 kg (7.56 mol) 
1-Hydroxybenzotriazole (HOBt) 
1.02 kg (7.55 mol) 
Boc-2-Aminoisobutyric acid (13) 
1.54 kg (7.58 mol) 
Isopropyl acetate 32 L 
1 M Aqueous sodium hydroxide 
38 L 
0.5 M Aqueous hydrochloric acid 
38 L 
Satd. aqueous sodium hydrogen carbonate 
38 L 
Absolute ethanol 45 L 
______________________________________ 
Water (49 L) was added to the stirred solution of the spiroindoline amine 
12 (3.057 kg) in isopropyl acetate (total volume .about.51 L) in a 
reaction vessel at room temperature under a nitrogen atmosphere. The 
following chemicals were then added sequentially: DCC (156 kg, .about.1.1 
equivs.), HOBt (1.02 kg, .about.1.1 equivs.) and finally, 
N-Boc-2-aminoisobutyric acid 13 (1.54 kg, .about.1.1 equivs.). The mixture 
was stirred vigorously at room temperature for 2 hours when LC showed the 
reaction to be complete. The mixture was filtered to to another vessel via 
an Estrella filter using a pump. Isopropyl acetate (22 L) was used to 
rinse vessel, the filter, the pump and the lines into the receiving 
vessel. The 2-phase mixture was then stirred for 5 minutes and the layers 
were allowed to separate. The lower aqueous layer was separated without 
incident (weight of aqueous liquors=51.1 kg). The organic solution was 
then washed sequentially with 1M aqueous sodium hydroxide (38 L), 0.5M 
aqueous hydrochloric acid (38 L) and finally, saturated aqueous sodium 
hydrogen carbonate (38 L) without incident. 
The organic solution was then transferred using a pump via an in-line 
filter (containing a 10.mu. cartridge) to another vessel for the solvent 
switch to ethanol. The vessel was rinsed with isopropyl acetate (10 L) and 
this was used to rinse the pump, the filter and the lines into the 
receiving vessel. The filtrate and washings were combined. Total volume=75 
L (by dipstick). LC assay gave 4.395 kg of Boc-aminoisobutyryl 
O-benzylserine spiroindoline (14), i.e. 93% overall from 7.49 moles of 
starting CBZ-spiroindoline sulfonamide (1). 
The batch was concentrated in vacuo to low bulk (-15 L) and the isopropyl 
acetate switched to ethanol by "feeding and bleeding" absolute ethanol (45 
L total). At the end of the solvent switch, GC showed &lt;1% isopropyl 
acetate remaining. This solution (25 L) containing 4.395 kg of 14 was used 
for the next stage. If desired, the inermediate compound 14 may be 
isolated by adding water to crystalize it out. 
EXAMPLE 17A 
##STR22## 
Aminoisobutyryl O-Benzylserine Spiroindoline (15) 
______________________________________ 
Materials: 
______________________________________ 
Boc Spiroindoline (14) 
3160 g (5.03 moles) 
Methanesulfonic acid (MsOH) 
979 mL (15.1 moles) 
EtOH 6.2 L 
H.sub.2 O 30 L 
1N NaOH 11 L 
EtOAc 26 L 
Darco 60 activated carbon 
1 Kg 
______________________________________ 
The Boc spiroindoline 14 was dissolved in 6.2 L of EtOH and treated with 
MsOH (979 mL). The temperature rose from 20 to 30.degree. C. and the 
reaction was allowed to proceed overnight. After 12 hours at 20.degree. C. 
there was still 15 A % of starting material left so the mixture was heated 
to 35.degree. C. for 6 hours. Upon completion (&lt;0.1 A % 14) the reaction 
was cooled to 200.degree. C. and 30 L of H.sub.2 O were added and the 
solution was filtered through a glass funnel with a polypropylene filter 
to filter off residual DCU. The mixture was transferred to a 100 L 
extractor and 26 L of EtOAc were added. The aqueous layer was basified via 
addition of chilled IN NaOH (11 L) and 1 L of 50% NaOH. Addition of ice 
was required to keep the temperature below 14.degree. C. Higher 
temperatures resulted in significant emulsion problems. 
The organic layer was distilled at 50.degree. C. at ca. 21" of Hg until 
KF&lt;1000 .mu.g/mL. Lower KF's result in more efficient carbon treatments 
and better recovery at the salt formation step. KF's of 160 .mu.g/mL were 
achieved at the 700 g scale. The solution was diluted with ethyl acetate 
to a total volume of 31 L (LC assay 2.40 kg). Activated carbon (Darco 
G-60) was added and the mixture was stirred for 24 h. The mixture was 
filtered through Solka Floc.TM. and the filter cake was washed with ethyl 
acetate (16 L), assay 2.34 Kg. 
EXAMPLE 17B 
##STR23## 
Aminoisobutyryl O-Benzylserine Spiroindoline (15) 
______________________________________ 
Materials: 
______________________________________ 
Boc Spiroindoline (14) 
4.395 kg (6.99 mol) 
Methanesulfonic acid 2.017 kg (20.99 mol) 
Ethyl acetate 185 L 
1 M Aqueous sodium hydroxide 
16 L 
50% Aqueous sodium hydroxide 
2.6 L 
Darco G-60 900 g 
Solka Floc .TM. 2.5 kg 
______________________________________ 
Methanesulfonic acid (2.017 kg, 1.36 L, .about.3 equivs.) was added to the 
stirred solution of the Boc spiroindoline 14 (4.395 kg) in ethanol (total 
volume .about.25 L) in a reaction vessel at room temperature. The batch 
was warmed to 35-40.degree. C., and stirred overnight. On the next day, 
the batch contained .about.1.1 A % of starting material and so the 
reaction was continued for a further 4 hours, then LC showed ratio of 
product/starting material to be 99.6/0.4. The batch was concentrated in 
vacuo to .about.15 L volume and then diluted with water (44 L). The batch 
was cooled to 5.degree. C., stirred for 30 minutes and then filtered 
through a Sparkler in-line filter (containing a 10.mu. cartridge) using a 
pump to another vessel to remove a small amount of residual DCU. 
The vessel, the pump, the filter and the lines were rinsed with water (10 
L), and this was added to the vessel. Ethyl acetate (36 L) was added to 
the vessel and the stirred mixture was cooled to 10.degree. C. A solution 
of cold (5-10.degree. C.) 1M aqueous sodium hydroxide solution (16 L) and 
cold (5-10.degree. C.) 50% aqueous sodium hydroxide solution (2.6 L) were 
added at 10.degree. C. and the temperature rose to 14.degree. C. The 
resulting mixture was stirred for 15 minutes at &lt;14.degree. C. and then 
the lower aqueous layer separated off. 
The batch was concentrated in vacuo to .about.20 L volume and then a 
mixture of ethyl acetate (35 L) and ethanol (5 L) was fed in while 
maintaining the volume at .about.20 L. At the end of this distillation the 
KF was 9160 mgml.sup.-1. The batch was solvent switched to ethyl acetate 
by "feeding and bleeding" ethyl acetate (40 L total). At the end of this 
distillation, KF was 446 mgml.sup.-1. The batch was diluted with ethyl 
acetate (10 L). 
Darco G-60 (900 g) was added to the hazy mixture. This was rinsed in with 
ethyl acetate (6 L). This mixture was stirred at room temperature 
overnight. Next day, Solka Floc.TM. (0.5 kg) was added to the stirred 
batch in the vessel and then Solka Floc.TM. (2.0 kg) was stirred in a 
little ethyl acetate and loaded into an Estrella filter. The excess 
solvent was pumped away through a Sparkler in-line filter containing a 
10.mu. cartridge. The slurry was transferred from the vessel through a 
filter using a pump and then through another filter to 2.times.40 L 
stainless steel bins. Visual inspection showed the liquors to be clear and 
clean. The vessel was rinsed with ethyl acetate (22 L) and this was used 
to rinse through the route outlined above to the stainless steel cans. The 
contents of both cans was transferred into a reaction vessel and the 
solution was mixed thoroughly. 
The batch (58 L) had a KF of 2950 mgml.sup.-1 and so was redried by 
concentrating in vacuo to 20-25 L volume. The batch was diluted to 46 L 
volume (dipstick) by the addition of ethyl acetate (25 L). The KF was 363 
mgml.sup.-1. The batch was diluted to 62 L volume by the addition of ethyl 
acetate (17 L) and was used for the final stage of the process. 
EXAMPLE 18A 
##STR24## 
Spiro[3H-i 
ndole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-m 
ethylpropanamide Methanesulfonate (16) 
______________________________________ 
Materials: 
______________________________________ 
Amine (15) 2340 g (4.43 moles) 
Methane sulfonic acid (MsOH) 
316 mL (4.88 moles) 
EtOAc 60 L 
EtOH 4.8 L 
8% EtOH in EtOAc 20 L 
______________________________________ 
The volume of the solution of 15 from the previous step was adjusted to 60 
L with ethyl acetate and EtOH (4.8 L) was added. The MsOH (316 mL) was 
added in 3 L of EtOAc at 45.degree. C. To the deep red homogeneous 
solution was added 496 g of the title compound Form I seed (10% seed based 
on the weight of the free amine was employed). The temperature rose to ca. 
48.degree. C. and the reaction was aged at 52.degree. C. for 1.5 hours. 
Analysis indicated complete conversion to the title compound (Form I). (At 
less than 10% seed longer age (&gt;3 hours) was required). The slurry was 
allowed to cool to 20.degree. C. overnight and was filtered in a 
centrifuge under N.sub.2. The cake was washed with 20 L of 8% EtOH in 
EtOAc. N.sub.2 is essential during filtration because the wet crystals are 
very hygroscopic. The batch was dried at 35.degree. C. under vacuum to 
afford 2.7Kg (56% overall yield) of the title compound (Form I) (99.9 A % 
purity; &lt;0.1% enantiomer). 
The conversion of Form II to Form I is also accomplished where the salt is 
formed in EtOAc-EtOH by addition of MsOH as above and the initial solution 
of the salt (at 55.degree. C.) is cooled to 45.degree. C. Crystals start 
appearing at that temperature and the slurry becomes thicker with time. 
The temperature is then raised to 51.degree. C. and the slurry is aged 
overnight. Complete conversion to Form I of 16 should be expected. This 
procedure may also be employed to prepare seed crystals of Form I of 16. 
EXAMPLE 18B 
##STR25## 
Spiro[3H-i 
ndole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-m 
ethylpropanamide Methanesulfonate (16) 
______________________________________ 
Materials: 
______________________________________ 
Amine (15) 3.1 kg (5.86 mol) 
Methanesulfonic acid 620 g (6.45 mol) 
Ethyl acetate 37 L 
Absolute ethanol 8.7 L 
Spiro[3H-indole-3,4'-piperdin]-1'-yl)- 
70 g (0.11 mol) 
carbonyl)-2-(phenylmethyl-oxy)ethyl]- 
2-amino-2-methylpropanamide 
methanesulfonate (Form I) 
______________________________________ 
Absolute ethanol (6.4 L) was added to the solution of the amine (15) (3.1 
kg) in ethyl acetate (total volume .about.62 L) in a reacttion vessel. The 
batch was warmed to 50.degree. C. and a solution of methanesulfonic acid 
(620 g, 412 ml, 1.1 equivs.) in ethyl acetate (11 L) was added over 
.about.5 minutes at 50-54.degree. C. The batch was seeded with 
spiro[3H-indole-3,4'-piperdin]-1'-yl)-carbonyl]-2-(phenylmethyl-oxy)ethyl] 
-2-amino-2-methylpropanamide methanesulfonate (Form I) (70 g) and the 
resulting slurry was stirred and heated at 55.degree. C. under nitrogen 
atmosphere overnight. 
The next day, the slurry was cooled to 15-20.degree. C., held for 2 hours 
and then dropped to the 50 cm polypropylene filter under nitrogen 
atmosphere. The solid product was washed with a mixture of absolute 
ethanol (2.3 L) in ethyl acetate (26 L). The white, solid product was dug 
off and dried in an Apex oven in vacuo at 35.degree. C. for an appropriate 
time (approx. two days). The dried 
spiro[3H-indole-3,4'-piperdin]-1'-yl)-carbonyl]-2-(phenylmethyl-oxy)ethyl] 
-2-amino-2-methylpropanamide methanesulfonate (3.352 kg) was sieved using a 
Jackson-Crockatt sieve to give 3.347 kg (including seed, 70 g)} 
yield=3.277 kg. 
HPLC Conditions: 
LC Retention times on Zorbax RX-C8 (4.6 mm.times.25 cm), .lambda.=210 nm, 
flow rate=1.5 ml/min. 
Compound 1: 60:40 CH.sub.3 CN--H.sub.2 O (1% H.sub.3 PO.sub.4) RT=5.0 min 
Compound 1b: 35:65 CH.sub.3 CN--H.sub.2 O (0.1 w % NH.sub.4 OAc) RT=6.2 
min. 
Compound 10: 60:40 CH.sub.3 CN--H.sub.2 O (0.1 H.sub.3 PO.sub.4) RT=2.9 
min. 
Compound 11: 60:40 CH.sub.3 CN--H.sub.2 O (0.1% H.sub.3 PO.sub.4) RT=5.4 
min. 
Compound 12: 40:60 CH.sub.3 CN--H.sub.2 O [pH 5.25 NaH.sub.2 PO4 (6.9 g/L 
of H.sub.2 O) (adjust pH with NaOH)] RT=5.6 min 
Compound 14: 60:40% CH.sub.3 CN--H.sub.2 O (0.1% H.sub.3 PO.sub.4) RT=4.65 
min 
Compound 15: 40:60% CH.sub.3 CN--H.sub.2 O [pH=5.25 NaH.sub.2 PO4 (6.9 g/L 
of H.sub.2 O)] adjust pH with NaOH)RT=4.9 min 
LC Retention times on Zorbax RX-C8 (4.6 mm.times.25 cm), .lambda.=210 nm, 
flow rate=1.2 ml/min, column temperature=48.degree. C. 
Solvent A=0.05% Phosphoric acid+0.01% Triethylamine in water 
Solvent B=Acetonitrile 
Gradient system: 
______________________________________ 
Time % A % B 
______________________________________ 
0 min 95 5 
35 min 10 90 
38 min 95 5 
40 min 95 5 
______________________________________ 
______________________________________ 
Retention time (mins) 
______________________________________ 
Compound 1 25.2 
Compound 1b 8.5 
Compound 10 20.5 
Compound 11 26.3 
Compound 12 14.8 
Compound 14 25.6 
Compound 15 15.7 
______________________________________ 
EXAMPLE 19 
Procedure for Manufacturing 1.0 mg Potency Tablets of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate 
______________________________________ 
Ingredient Per Tablet 
Per 2600 Tablets 
______________________________________ 
Active ingredient (N-[1(R)-[(1,2-dihydro- 
1.18 mg 3.068 g 
1-methane-sulfonylspiro[3H-indole-3,4'- 
piperdin]-1'-yl)carbonyl]-2-(phenyl- 
methyloxy)-ethyl]-2-amino-2-methyl- 
propanamide methanesulfonate) 
Calcium Phosphate Dibasic 
47.32 mg 123.03 
g 
Starch Pregelatinized NF 1500 
30.00 mg 78.0 g 
Microcrystalline Cellulose NF Avicel PH 
15.00 mg 39.0 g 
101 
Magnesium Stearate Impalpable Powder 
0.50 mg 1.3 g 
NF 
Croscarmellose Sodium NF 
12.75 mg 33.15 g 
Ethanol 95% 7.5 .mu.l 19.5 ml 
Water purified (Tablet Weight = 100 g) 
22.5 .mu.l 58.5 ml 
______________________________________ 
The active ingredient (equivalent to 1.0 mg anhydrous free base per tablet) 
was mixed with the calcium phosphate dibasic, the starch pregelatinized NF 
1000, the microcrystalline cellulose NF, and half of the croscarmellose 
sodium NF in a high shear granulator for 5 minutes. The 25% ethanol/water 
granulating solution was slowly added to the powder mixture with the mixer 
running over a period of about 1.5 minutes then granulated for about 7 
minutes to form granules. The wet granules were dried at about 47.degree. 
C. (range 46 to 48.degree. C.) in a tray dryer or a fluid bed dryer for 
approximately 3.0 hours. The dried granules were then milled using a 
Quadro Comill to achieve fine granules. After milling, the remainder of 
the croscarmellose sodium NFS was added to the fine granules and mixed in 
a V blender for about 10 minutes. Magnesium stearate impalpable powder NF 
was added to this blend through a 60 mesh stainless steel screen and 
blended in the V blender for about 1 minute. The lubricated mixture was 
compressed to provide tablets of 1.0 mg active ingredient (free base 
equivalent). 
EXAMPLE 20 
Procedure for Manufacturing 1.0 mg Potency Coated Tablets of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate 
______________________________________ 
Ingredient Per Tablet 
Per 2600 Tablets 
______________________________________ 
Hydroxypropyl Methylcellulose USP 
0.80 mg 2.08 g 
(HPMC) 
Hydroxypropyl Cellulose NF with &lt;0.3% 
0.80 mg 2.08 g 
Silica (HPC) 
Titanium Dioxide USP 
0.32 mg 0.83 g 
Talc USP Purified 0.08 mg 0.21 g 
Water Purified (Film Coated Tablet 
To 20 .mu.l To 52 ml 
Weight = 102 g) 
______________________________________ 
The titanium dioxide and talc, USP were mixed and passed through a 60 mesh 
stainless steel screen. This mixture was mixed with HPMC and HPC to form a 
dry blend. The dry blend was added to water (20 ml) which was previously 
heated to 90.degree. C. with mild agitation to ensure that the blend is 
wetted to form a slurry. The remainder of the water (up to 32 ml) was 
added to the slurry at ambient temperature with gentle agitation to form a 
suspension. The suspension was then applied to the tablets from the 
previous Example using the following guidelines to provide the coated 
tablets. 
Pan: suitable size 
Pan Speed: 20 RPM 
Nozzles: 2850 liquid/120 air 
Inlet Temperature: 85.degree. C. 
Bed Temperature: 47.degree. C. 
Spray Rate: ca. 2.0 g/minute/kg Tablets 
EXAMPLE 21 
Procedure for Manufacturing 5.0 mg Potency Tablets of 
N-[1(R)[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl 
)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate 
______________________________________ 
Ingredient Per Tablet 
Per 25,000 Tablets 
______________________________________ 
Active Ingredient (N-[1(R)-[1,2- 
5.91 mg 147.8 g 
dihydro-1-methane-sulfonylspiro- 
[3H-indole-3,4'-piperdin]-1'-yl)- 
carbonyl]-2-(phenylmethyloxy)- 
ethyl]-2-amino-2-methylpropan- 
amide methanesulfonate) 
Calcium Phosphate Dibasic 
188.10 mg 4.70 kg 
Starch Pregelatinized NF 1500 
120.00 mg 3.00 kg 
Microcrystalline Cellulose NF Avicel 
60.00 mg 1.50 kg 
PH 101 
Magnesium Stearate Impalpable 
2.00 mg 50.0 g 
Powder NF 
Croscarmellose Sodium NF 
24.00 mg 600 g 
Ethanol 95% 30 .mu.l 750 ml 
Water purified (Tablet Weight = 
90 .mu.l 2.25 l 
400 g) 
______________________________________ 
The active ingredient (equivalent to 5.0 mg anhydrous free base per tablet) 
was mixed with the calcium phosphate dibasic, the starch pregelatinized NF 
1000, the microcrystalline cellulose NF, and half of the croscarmellose 
sodium NF in a high Fielder 10/25 mixer for about 6 minutes. The 25% 
ethanol/water granulating solution was slowly added to the powder mixture 
with the mixer running over a period of about 1.5 minutes then granulated 
for about 8 minutes to form granules. The wet granules were dried at about 
47.degree. C. (range 46 to 48.degree. C.) in a tray dryer or a fluid bed 
dryer for approximately 3.0 hours. The dried granules were then milled 
using a Quadro Comill to achieve fine granules. After milling, the 
remainder of the croscarmellose sodium NFS was added to the fine granules 
and mixed in a V blender for about 10 minutes. Magnesium stearate 
impalpable powder NF was added to this blend through a 60 mesh stainless 
steel screen and blended in the V blender for about 1 minute. The 
lubricated mixture was compressed to provide tablets of 5.0 mg active 
ingredient (free base equivalent). 
EXAMPLE 22 
Procedure for Manufacturing 5.0 mg Potency Coated Tablets of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate 
______________________________________ 
Ingredient Per Tablet 
Per 25,000 Tablets 
______________________________________ 
Hydroxypropyl Methylcellulose USP 
3.2 mg 80 g 
(HPMC) 
Hydroxypropyl Cellulose NF with 
3.2 mg 80.0 g 
&lt;0.3% Sillca (HPC) 
Titanium Dioxide USP 
1.28 mg 32.0 g 
Talc USP Purified 0.32 mg 8.0 g 
Water Purified (Film Coated Tablet 
To 80 .mu.l To 200 ml 
Weight = 408 g) 
______________________________________ 
Using essentially the procedure of the prior Example 20 and applying the 
suspension to the tablets from the previous Example, 5.0 mg potency coated 
tablets were formed. 
EXAMPLE 23 
Procedure for Manufacturing 25 mg Potency Tablets of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate 
______________________________________ 
Ingredient Per Tablet 
Per 25,000 Tablets 
______________________________________ 
Active Ingredient (N-[1(R)-[(1,2- 
29.55 mg 738.75 g 
dihydro-1-methane-sulfonylspiro- 
[3H-indole-3,4'-piperdin]-1'-yl)- 
carbonyl]-2-(phenylmethyloxy)- 
ethyl]-2-amino-2-methylpropanamide 
methanesulfonate) 
Calcium Phosphate Dibasic 
174.46 mg 4.361 kg 
Starch Pregelatinized NF 1500 
113.00 mg 2.825 kg 
Microcrystalline Cellulose NF Avicel 
57.00 mg 1.425 kg 
PH 101 
Magnesium Stearate Impalpable 
2.00 mg 50.0 g 
Powder NF 
Croscarmellose Sodium NF 
24.00 mg 600 g 
Ethanol 95% 30 .mu.l 750 ml 
Water purified (Tablet Weight = 
90 .mu.l 2.25 l 
400 g) 
______________________________________ 
The active ingredient (equivalent to 25 mg anhydrous free base per tablet) 
was mixed with the calcium phosphate dibasic, the starch pregelatinized NF 
1000, the microcrystalline cellulose NF, and half of the croscarmellose 
sodium NF in a high shear granulator Fielder 10/25 mixer for about 6 
minutes. The 25% ethanol/water granulating solution was slowly added to 
the powder mixture with the mixer running over a period of about 1.5 
minutes then granulated for about 8 minutes to form granules. The wet 
granules were dried at about 47.degree. C. (range 46 to 48.degree. C.) in 
a tray dryer or a fluid bed dryer for approximately 3.0 hours. The dried 
granules were then milled using a Quadro Comill to achieve fine granules. 
After milling, the remainder of the croscarmellose sodium NFS was added to 
the fine granules and mixed in a V blender for about 10 minutes. Magnesium 
stearate impalpable powder NF was added to this blend through a 60 mesh 
stainless steel screen and blended in the V blender for about 1 minute. 
The lubricated mixture was compressed to provide tablets of 25 mg active 
ingredient (free base equivalent). 
EXAMPLE 24 
Procedure for Manufacturing 25 mg Potency Coated Tablets of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate 
______________________________________ 
Ingredient Per Tablet 
Per 25,000 Tablets 
______________________________________ 
Hydroxypropyl Methylcellulose USP 
3.2 mg 80 g 
(HPMC) 
Hydroxypropyl Cellulose NF with 
3.2 mg 80.0 g 
&lt;0.3% Silica (HPC) 
Titanium Dioxide USP 
1.28 mg 32.0 g 
Talc USP Purified 0.32 mg 8.0 g 
Water Purified (Film Coated Tablet 
To 80 .mu.l To 200 ml 
Weight = 408 g) 
______________________________________ 
Using essentially the procedure of Example 20 and applying the suspension 
to the tablets from the previous Example, 25 mg potency coated tablets 
were formed. 
EXAMPLE 25 
Procedure for Manufacturing 100 mg Potency Tablets of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate 
______________________________________ 
Ingredient Per Tablet 
Per 2600 Tablets 
______________________________________ 
Active Ingredient (N-[1(R)-[(1,2- 
118.20 mg 307.3 g 
dihydro-1-methane-sulfonylspiro[3H- 
indole-3,4'-piperdin]-1'-yl)-carbonyl]- 
2-(phenylmethoxyloxy)-ethyl]-2-amino- 
2-methylpropanamide methane- 
sulfonate) 
Calcium Phosphate Dibasic 
81.80 mg 212.7 g 
Starch Pregelatinized NF 1500 
78.00 mg 202.8 g 
Microcrystalline Cellulose NF Avicel 
60.00 mg 156.0 g 
PH 101 
Magnesium Stearate Impalpable Powder 
2.00 mg 5.20 g 
NF 
Croscarmellose Sodium NF 
60.00 mg 156.0 g 
Ethanol 95% 30.0 .mu.l 78.0 ml 
Water purified (Tablet Weight = 400 g) 
90.0 .mu.l 234.0 ml 
______________________________________ 
The active ingredient (equivalent to 100 mg anhydrous free base per tablet) 
was mixed with the calcium phosphate dibasic, the starch pregelatinized NF 
1000, the microcrystalline cellulose NF, and half of the croscarmellose 
sodium NF in a high shear granulator for 5 minutes. The 25% ethanol/water 
granulating solution was slowly added to the powder mixture with the mixer 
running over a period of about 1.5 minutes then granulated for about 7 
minutes to form granules. The wet granules were dried at about 47.degree. 
C. (range 46 to 48.degree. C.) in a tray dryer or a fluid bed dryer for 
approximately 3.0 hours. The dried granules were then milled using a 
Quadro Comill to achieve fine granules. After milling, the remainder of 
the croscarmellose sodium NFS was added to the fine granules and mixed in 
a V blender for about 10 minutes. Magnesium stearate impalpable powder NF 
was added to this blend through a 60 mesh stainless steel screen and 
blended in the V blender for about 1 minute. The lubricated mixture was 
compressed to provide tablets of 100 mg active ingredient (free base 
equivalent). 
EXAMPLE 26 
Procedure for Manufacturing 100 mg Potency Coated Tablets of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate 
______________________________________ 
Ingredient Per Tablet 
Per 2600 Tablets 
______________________________________ 
Hydroxypropyl Methylcellulose USP 
3.2 mg 8.32 g 
(HPMC) 
Hydroxypropyl Cellulose NF with 
3.2 mg 8.32 g 
&lt;0.3% Silica (HPC) 
Titanium Dioxide USP 
1.28 mg 3.33 g 
Talc USP Purified 0.32 mg 0.83 g 
Water Purified (Film Coated Tablet 
To 80.0 .mu.l To 208 ml 
Weight = 408 g) 
______________________________________ 
Using essentially the procedure of Example 20 and applying the suspension 
to the tablets from the previous Example, 100 mg potency coated tablets 
were formed. 
EXAMPLE 27 
Preparation of amorphous form of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate 
As a mimic of the tablet formulation process, a concentrated solution of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate (118 mg) in 120 .mu.L 25% aqueous ethanol (980 mg/ml) was 
evaporated at 40.degree. C. to give a solid. The lack of crystallinity was 
confirmed by X-ray analysis. The X-ray diffraction pattern showed an 
amorphous halo. Examination of the solid under microscopy showed no 
biorefringence. 
The solid state chemical stability of the amorphous form was studied after 
12 weeks at 40.degree. C., 60.degree. C. and 80.degree. C., and it was 
found to exhibit excellent stability. After 12 weeks at 40.degree. C., 
100% of the initial compound was present; after 12 weeks at 60.degree. C., 
99.7% of the initial compound was present; after 12 weeks at 80.degree. 
C., 97.8% of the initial compound was present. 
EXAMPLE 28 
Chemical Stability of Film Coated Tablets of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate Following 6.0 Months Stability Study 
The results of a 6.0 month stability study of film coated tablets of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate are presented below. 
______________________________________ 
% of initial 
% of initial 
Stability Chamber 1.0 mg dose 
100 mg dose 
______________________________________ 
30.degree. C./Amient Humidity 
99 99 
30.degree. C./75% Relative Humidity 
99 99 
40.degree. C./75% Relative Humidity 
99 99 
______________________________________ 
In particular, no degradates were observed for the 100 mg dose. Degradates 
were observed for the 1.0 mg dose varying from 0.1 to 0.7 area % relative 
to active only at 40.degree. C./75% relative humidity. Furthermore, the 
tablet dissolution, disintegration, and hardness for both the 1.0 mg dose 
and the 100 mg dose were satisfactory following 6.0 months storage under 
the above conditions. 
EXAMPLE 29 
Tablet Crushing Strength and Disintegration Times of 100 mg Potency Tablets 
of N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro 
[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amin 
o-2-methylpropanamide methanesulfonate 
Tablets of 400 mg compression weight were prepared by the procedures of the 
above examples using 118.2 mg of 
N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-y 
l)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide 
methanesulfonate per tablet (29.6% tablet weight). All granulations were 
prepared on a several gram scale by pestle and morter using 25% 
ethanol/75% water as a granulating solution. Tablets were compressed on a 
Carver Press under 1000 lb force (uncoated tablet cores), unless otherwise 
noted. 
Formulation A: wet granulation--Starch Pregelatinized NF 1500 (34.8%); 
Microcrystalline Cellulose NF Avicel PH 101 (34.8%); Talc USP (0.6%); 
Magnesium Stearate (0.3%). 
Formulation B: direct compression--Calcium Phosphate Dibasic (26.3%); 
Microcrystalline Cellulose NF Avicel PH 102 (39%); Talc USP (1.2%); 
Magnesium Stearate (0.6%); Croscarmellose Sodium NF (3%). 
Formulation C: wet granulation, compressed under 500 lb force--Starch 
Pregelatinized NF 1500 (33.3%); Microcrystalline Cellulose NF Avicel PH 
101 (33.3%); Talc USP (0.6%); Magnesium Stearate (0.3%); Croscarmellose 
Sodium NF (3%=1.5% intragranular+1.5% extragranular). 
Formulation D: wet granulation--Starch Pregelatinized NF 1500 (33.3%); 
Microcrystalline Cellulose NF Avicel PH 101 (33.3%); Talc USP (0.6%); 
Magnesium Stearate (0.3%); Croscarmellose Sodium NF (3% extragranular). 
Formulation E: wet granulation--Microcrystalline Cellulose NF Avicel PH 101 
(36.4%); Calcium Phosphate (26.3%); Talc USP (1.2%); Magnesium Stearate 
(0.6%); Croscarmellose Sodium NF (6%=3% intragranular+3% extragranular). 
Formulation F: wet granulation--Microcrystalline Cellulose NF Avicel PH 101 
(15.0%); Calcium Phosphate (29.5%); Starch Pregelatinized NF 1500 (19.5%); 
Magnesium Stearate (0.5%); Croscarmellose Sodium NF (6%=3% 
intragranular+3% extragranular). 
Formulation G: wet granulation--Microcrystalline Cellulose NF Avicel PH 101 
(15.0%); Calcium Phosphate (26.5%); Starch Pregelatinized NF 1500 (19.5%); 
Magnesium Stearate (0.5%); Croscarmellose Sodium NF (9%=3% 
intragranular+6% extragranular). 
Formulation H: wet granulation--Microcrystalline Cellulose NF Avicel PH 101 
(15.0%); Calcium Phosphate (26.5%); Starch Pregelatinized NF 1500 (19.5%); 
Magnesium Stearate (0.5%); Croscarmellose Sodium NF (9%=3% 
intragranular+6% extragranular); Super Disintegrant (12% extragranular). 
______________________________________ 
Formulation 
Tablet Hardness (kP) 
Disintegration Time (min) 
______________________________________ 
A Does not break 13.0 
B 23.3 (one tablet) 
7.0 
C 23.7 (one tablet) 
14.0 
D 29.2 .+-. 1.2 (three tablets) 
13.5 .+-. 0.25 
E 14.1 .+-. 0.7 (three tablets) 
12.5 .+-. 1.6 
F 13.5 .+-. 1.1 (three tablets) 
20.5 .+-. 0.0 
G 18.5 (two tablets) 
14.0 
H 14.9 (two tablets) 
7.5 
______________________________________ 
As demonstrated above, the present formulations have superior properties 
regarding strength and stability. The tablet hardness is suitable for film 
coating and disintegration time is not too long. 
While the invention has been described and illustrated with reference to 
certain particular embodiments thereof, those skilled in the art will 
appreciate that various adaptations, changes, modifications, 
substitutions, deletions, or additions of procedures and protocols may be 
made without departing from the spirit and scope of the invention. For 
example, effective dosages other than the particular dosages as set forth 
herein above may be applicable as a consequence of variations in the 
responsiveness of the mammal being treated for any of the indications with 
the compound of the invention indicated above. Likewise, the specific 
pharmacological responses observed may vary according to and depending 
upon the particular active compound selected or whether there are present 
pharmaceutical carriers, as well as the type of formulation and mode of 
administration employed, and such expected variations or differences in 
the results are contemplated in accordance with the objects and practices 
of the present invention. It is intended, therefore, that the invention be 
defined by the scope of the claims which follow and that such claims be 
interpreted as broadly as is reasonable.