Patent Application: US-24202699-A

Abstract:
disclosed are conjugates of a polypeptide or oligopeptide with a low molecular weight lipophilic compound , the peptide and lipophilic group being covalently linked to one another either directly or via a connecting element , with the exception of conjugates in which the peptide component consists of hirudin or peptide structurally derived from hirudin or a peptide derivative with hirudin - like properties , and conjugates whose peptide component has been hydrophobically modified during their biosynthesis .

Description:
the present invention relates to prodrugs designed according to a novel concept for developing better routes of drug administration and consequent enhanced drug stability and bioactivity . according to the novel approach of the present invention , it is preferred to lose rather than to preserve the drug &# 39 ; s native structure , biological potency and target - recognition capacity but , following application , the thus modified drug will spontaneously and slowly convert back in the body to the original active molecule . according to the novel concept of the present invention , numerous currently applied drugs can be converted into inactive prodrugs that are long - lived species as they evade general and receptor - mediated degradation in the organism . the prodrugs of the invention are designed to undergo spontaneous regeneration into the original drugs under in vivo physiological conditions and in a homogeneous fashion . the wide spectrum of chemical procedures available for the production of the prodrugs of the invention allow to obtain a rapid or slow rate of reactivation as necessary . thus given prodrugs may be prepared with physical attributes such as decreased solubility and , therefore , a slower rate of s . c . absorption . in addition , altering the hydrophobicity index of prodrugs , combined with the feature of spontaneous regeneration in blood circulation , permits to convert orally nonabsorptive drugs into gastrointestinal permeable prodrugs . the prodrugs according to the invention include modified drugs for use in humans and animals as well as modified insect pheromones . in one aspect of the invention the prodrug is modified insulin . at present , insulin is the predominant drug for diabetes mellitus , a group of syndromes characterized by hyperglycemia , altered metabolism of lipids , carbohydrates and proteins and an increased risk of complications from vascular diseases . most patients can be classified clinically as having either insulin - dependent ( iddm , type i ) or insulin - independent diabetes mellitus ( niddm , type ii ). in the western world about 90 % of diabetic patients have type ii diabetes and most of the remainder have type i . about 70 % of type ii diabetics in the united states are also obese , a factor that contributes significantly to insulin resistance . in type i diabetes , there is an extensive and selective loss of pancreatic β - cells and a state of hypoinsulinemia . by contrast there is no significant loss of β - cells from the islets in type ii diabetic patients , in which patients the mean plasma concentration of insulin over a 24 - hour period is essentially normal or even elevated because of peripheral resistance to the action of the hormone . nevertheless , individuals with type ii diabetes are relatively insulin deficient . this is because a normal pancreatic β - cell should be capable of secreting amounts of insulin that are considerably greater than normal when confronted with hyperglycemia , thus allowing an individual to maintain euglycemia in the face of moderate resistance to insulin . virtually all forms of diabetes mellitus are due to either a decrease in the circulating concentration of insulin ( insulin deficiency ) or a decrease in response of peripheral tissues to insulin ( insulin resistance ), in association with an excess of hormones with actions opposite to those of insulin ( glucagon , growth hormone , cortisol , and catecholamines ). these hormonal abnormalities lead to alterations in the metabolism of carbohydrates , lipids , ketones and amino acids . the central feature of the syndrome is hyperglycemia . the half - life of insulin in plasma is about 5 - 6 min . degradation of insulin occurs primarily in liver and to a lesser extent in kidney and muscle . about 50 % of the insulin that reaches the liver in the portal vein is destroyed and never reaches the general circulation . insulin is filtered by the renal glomeruli and is reabsorbed by the tubules which also degrade it . proteolytic degradation of insulin in the liver is primarily receptor mediated . following insulin receptor - binding the complex is internalized into small vesicles termed endosomes where degradation is initiated . some insulin is also delivered to lysosomes for degradation . in hepatocytes about 50 % of internalized insulin is degraded . insulin is critical for the management of diabetes ketoacidosis , and important in the treatment of hyperglycemic non - ketonic coma , and in the perioperative management of both type i and type ii diabetic patients . subcutaneous ( s . c .) administration of insulin is the primary treatment for all type i patients and most type ii diabetic patients who are not adequately controlled by diet and / or oral hypoglycemic agents . in all cases the goal is to normalize , not only blood glucose , but also all other aspects of the unbalanced metabolism resulting from hypoinsulinemia and hyperglycemia . long term treatment based primarily on s . c . administration of insulin does not mimic the normal rapid rise and decline of insulin secretion in response to injected nutrients , and possesses preferential peripheral rather than hepatic effects of insulin , but nevertheless considerable success has been achieved with this treatment . insulin preparations traditionally utilized for s . c . application are classified according to their duration of action into short , intermediate or long - acting insulins , and according to the species origin . human insulin is now widely available , and in theory , expected to be less immunogenic than porcine or bovine insulins as the last two differ from human insulin by one and three amino acids , respectively . however , when highly purified , all three insulins have low but measurable capacity to stimulate the immune response . preparations at neutral ph values are in general stable and permit storage for long periods of time at room temperature . for traditional and therapeutic purposes , doses and concentrations of insulin are expressed in units ( u ), based on the amount required to induce normoglycemia in fasting rabbits . homogeneous preparations of insulin contain about 25 u / mg . almost all commercial preparations of insulin are supplied in solution at a concentration of 100 u / ml . short or rapid - acting insulins are soluble preparations of crystalline zinc insulin dissolved in a neutral ph buffer , usually injected 30 - 45 minutes before meals . these preparations have the most rapid onset of action and the shortest duration . under stable metabolic conditions regular insulins are usually administered together with intermediate , or long - acting preparations . intermediate - acting insulins were designed to be less soluble in aqueous solutions , therefore they dissolve more gradually upon subcutaneous administration and their duration of action is longer . two preparations most frequently used are nph insulin ( nph stands for neutral protamine hagedorn ), a suspension of zinc - insulin crystals in phosphate buffer modified by the addition of protamine sulfate , and lente insulin , a suspension of insulin in acetate buffer modified by the addition of zinc chloride to minimize solubility of insulin . since short - acting insulin is expected to cover a period of 0 . 4 - 7 hrs and intermediate - acting insulin may cover a range of 1 . 5 - 20 hrs , the correct timing and doses of the combination of the two insulins must take into consideration the variable parameters such as nutritional behavior , night ( fasting ) hypoglycemia , and morning hyperglycemia , when the activity of the counter - regulatory hormones ( to insulin ) is increased . if rapid - acting and long or intermediate - acting insulin preparations are coadministered together , a common disadvantage of such combinations is that , upon mixing , some of the rapid - acting insulins can be complexed with excess zn 2 + or protamine of the long or intermediate - acting preparation , being thus converted into an intermediate and even long - acting insulin . long - acting insulins , such as ultralente insulin or extended zinc - insulin or protamine - zinc - insulin suspensions , are insulin preparations in which zinc , or zinc plus protamine , was added in excess in order to achieve insoluble preparations of insulin . they are suspensions of minute particles of zinc - insulin and differ only in the particle size which determines the duration of their action . unlike regular insulin , ultralente insulins have a very slow onset and a prolonged (“ flat ”) peak of action . they advocate to provide a low basal concentration of insulin throughout the day , but their long half - life makes it difficult to determine the optimal dosage and several days of treatment are required before a steady - state concentration can be achieved . bovine and porcine ultralente insulin have more prolonged course of action than does human ultralente insulin . it is recommended to initiate the therapy with three times the normal daily dose as a loading dose , followed by once or twice daily injections . insulin has three amino and six carboxyl groups available for modification . the insulin derivatives of the invention are substituted at the insulin a and b - chains by one or more of the radicals ( i ) to ( iv ) above at one or more of the terminal amino groups of the gly a1 and phe b1 radicals , at the ε - amino groups of the lys b29 , at the terminal carboxyl groups of asn a21 and thr b30 and / or at the free carboxyl groups of glu a4 , glu a17 , glu b13 , and glu b21 . in addition , the thus substituted carboxyl and / or amino insulin derivatives may be further substituted by one or more of the functional groups ( i ) to ( iv ) at one or more of the free hydroxyl groups of the residues thr a8 , ser a9 , ser a12 , tyr a14 , tyr a19 , ser b9 , tyr b16 , tyr b26 , thr b27 and thr b30 . the amino acid residue numbering used in the present specification and claims is that of the human insulin used in the examples . in one preferred embodiment of the present invention , the insulin derivatives are substituted by one or more fmoc moieties at the free terminal amino groups of gly a1 and phe b1 , and / or at the ε - amino group of lys b29 , thus obtaining insulin derivatives having 1 to 3 fmoc substituents at the a 1 , b 1 and / or b 29 positions of the insulin molecule , in particular gly a1 - n -( fmoc ) 1 -, phe b1 - n -( fmoc ) 1 - and lys b29 - n -( fmoc ) 1 - insulin , gly a1 , phe b1 - n -( fmoc ) 2 -, gly a1 , lys b29 - n -( fmoc ) 2 - and phe b1 , lys b29 - n -( fmoc ) 2 - insulin , and gly a1 , phe b1 , lys b29 - n -( fmoc ) 3 - insulin . reaction of insulin with activated fmoc , for example with 9 - fluorenylmethyl - n - hydroxysuccinimide ( fmoc - osu ) yields mono -, di - and tri - n - fmoc - insulins that can be readily resolved by hplc procedures and individually obtained in pure form . in order to obtain a di - n - fmoc - insulin as the sole product , one of the free amino groups is first protected , e . g . with the t - boc group , the protected insulin derivative is reacted with excess of fmoc - osu , and the protecting group is then removed , resulting in the desired di - n - fmoc - insulin . when administered to diabetic patients , the mono -, di - and tri - n - fmoc - are converted to native insulin in vivo leading to antidiabetic effects over various , including prolonged , periods of time . for substitution only of the carboxyl groups of insulin ( c - fm ), i . e . in the terminal asn a21 and thr b30 and in the glu a4 , glu a17 , glu b13 and glu b21 residues , the free amino groups of the insulin molecule are first protected , e . g . with t - boc groups , and then a reaction of 3 steps is carried out , wherein ( 1 ) the free carboxyl groups are converted into active ester groups by reaction , e . g . with o - nitrophenol or n - hydroxy - succinimide ; ( 2 ) reaction of the activated ester groups with 9 - fluorenylmethanol is performed in the presence of imidazole ; and ( 3 ) the protecting t - boc groups are removed . an alternative procedure involves a one - step direct esterification of carboxylic groups with n , n ′- dicyclohexylcarbodiimide , 9 - fluorenylmethanol and 4 - dimethylaminopyridine , followed by removal of t - boc groups . when fmoc - insulin derivatives substituted both at the amino and carboxyl groups ( n - fmoc , c - fm ) are desired , the n - fmoc derivative is first prepared with fmoc - osu and the n - fmoc insulin is then converted to active esters followed by reaction with 9 - fluorenylmethanol , as described above . for preparation of fm , fmoc - insulin derivatives substituted at the carboxyl and hydroxyl functions ( c - fm , o - fmoc ), the amino groups are first protected with t - boc , the c - fm insulin derivative is prepared as above , followed by reaction with 9 - fluorenylmethoxycarbonyl chloride and removal of the protecting n - t - boc groups . for preparation of fm , fmoc - insulin derivatives substituted at the amino , carboxyl and hydroxyl functions ( n , o - fmoc , c - fm ), the n - fmoc , c - fm insulin is prepared as above and is then reacted with 9 - fluorenylmethoxycarbonyl chloride . the modified insulins according to the invention may be prepared from any insulin suitable for human use , such as native , recombinant or mutated human , porcine or bovine insulin . examples of mutated insulins are the b16 - tyr → his human insulin analog ( kaarsholm and ludvigsen , 1995 ) and the lys b28 pro b29 human insulin analog ( insulin lispro ), in which the naturally occurring amino acid sequence at positions b28 and b29 is reversed . insulin lispro is equipotent to human insulin and yet absorbed more quickly from subcutaneous injection sites ( campbell et al ., 1996 ). in a preferred embodiment , the insulin is human insulin , either native or recombinant . the mono - n - fmoc - insulin derivatives of the present invention have 40 - 80 % of the native insulin biological potency , as determined by the [ poly ( glu 4 tyr )] seq id no : 1 phosphorylating assay . the di - and tri - n - fmoc - insulin derivatives have 2 - 9 % and & lt ; 1 % the of the native insulin biological potency , respectively , as determined by the [ poly ( glu4tyr )] seq id no : 1 phosphorylating assay . when properly used , in the right proportions , these three prototypes may in principle substitute the traditional mixtures of rapid , intermediate and long - acting insulins currently applied in subcutaneous therapy of diabetes . in the aspect related to insulin , the invention provides pharmaceutical compositions comprising one or more insulin derivatives of the invention and a pharmaceutically acceptable carrier . for long - acting effect , the composition will preferably comprise either the n -( fmoc ) 3 - insulin or the n -( fmoc ) 2 - insulin derivative alone or a mixture of both . the pharmaceutical composition may be presented in any suitable form , for example as an oral formulation or for subcutaneous injection . in another embodiment of this same aspect , the invention relates to a method for treatment of diabetes which comprises administering to a diabetic patient an effective dose of one or more insulin derivatives of the invention . in preferred embodiments , the derivative is either native or recombinant human n -( fmoc ) 3 - insulin or n -( fmoc ) 2 - insulin , or a mixture of both , administered at 5 - 8 day intervals . if necessary , the treatment with the fmoc - insulin derivative is completed by daily administration of rapid - acting insulin . for long - term treatment , insulin is mainly administered by subcutaneous injections . current treatment with long - acting insulins administered subcutaneously suffers from large variations in absorption among individual diabetic patients , due to the fact that the added substances can potentially diffuse away at the site of the subcutaneous injection . the present invention provides insulin derivatives with ‘ built - in ’ decreased solubility within the insulin molecule itself . this is expected to eliminate in humans the large variation in subcutaneous absorption and to minimize or even to eliminate interferences upon mixing the analogs as well . the proper mixture of the three n -( fmoc )- insulin prototypes may cover a prolonged duration of action as it combines the need for rapid - acting insulin , together with the slow - released effects of n -( fmoc ) 2 and n -( fmoc ) 3 - insulin , all of which can be mixed together with no interfering effects . in another embodiment of this same aspect , the composition of the invention comprises a mixture of mono , di and tri - n - fmoc - insulin derivatives . n -( fmoc ) 3 - insulin is basically a long - acting insulin , n -( fmoc ) 1 - insulins are 40 - 80 % biologically active , as determined by the [ poly ( glu 4 tyr )] seq id no : 1 phosphorylating assay , and exhibit higher solubility in aqueous solutions , and n -( fmoc ) 2 - insulins are 2 - 9 % biologically active , as determined by the [ poly ( glu 4 tyr )] phosphorylating assay , and are less soluble in aqueous solutions . all three analogs return to fully active insulins upon incubation at 37 ° c . at physiological ph values . thus , the proper mixture of the three analogs may give rapid , intermediate and long - acting effect , presently achieved by multiple injections of regular insulin together with preparations containing zinc and protamine . in other aspects of the present invention , the prodrugs are derived from drugs for human or veterinary use including , but not being limited to , antidiabetic , antiinflammatory , antibacterial , antiviral , antineoplastic and antihypertensive drugs , as well as drugs for treatment of immunological , dermatological and neurological disorders . the pharmaceutical compositions of the invention comprise the prodrug or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier . any suitable route of administration of drugs to humans and animals is envisaged by the invention , for example via conventional injectable , implantable , oral , rectal or topical administration . these preparations can be prepared by conventional methods known to those skilled in the art , for example as described in “ remington &# 39 ; s pharmaceutical science ”, a . r . gennaro , ed ., 17th edition , 1985 , mack publishing company , easton , penn ., usa . the invention will now be illustrated by the following non - limiting examples . male wistar rats ( 1 80 - 200 g ) were supplied by the department of hormone research , weizmann institute of science . diabetes was induced by a single intravenous injection of a freshly prepared solution of streptozotocin ( 55 mg / kg body weight ) in 0 . 1m citrate buffer ( ph 4 . 5 ) according to meyerovitch et al ., 1987 . ( ii ) enriched preparation of insulin receptor tyrosine kinase was obtained from rat liver membranes as described by meyerovitch et al ., 1990 . briefly , the liver was homogenized in the presence of proteinase inhibitors , solubilized with 1 % triton x - 100 , and centrifuged . the supernatant was allowed to pass through a wheat - germ agglutinin ( wga )- agarose column ( sigma ). adsorbed insulin receptor portion was eluted with 0 . 3m n - acetyl - d - glucose amine in 50 mm hepes buffer , ph 7 . 4 , containing 0 . 1 % triton x - 100 , 10 % glycerol , and 0 . 15m nacl . biological potencies of insulin and insulin derivatives were evaluated by the assays ( iii ) and ( iv ) below . ( iii ) lipogenesis ( incorporation of labeled glucose into the lipids of intact adipocytes ) rat adipocytes were prepared essentially by the method of rodbell , 1964 . the fat pads of male wistar rats were cut into small pieces with scissors and suspended in 3 ml of krb buffer containing nacl , 110 mm ; nahco 3 , 25 mm ; kcl , 5 mm ; kh 2 po 4 , 1 . 2 mm ; cacl 2 , 1 . 3 mm ; mgso 4 , 1 . 3 mm ; and 0 . 7 % bsa ( ph 7 . 4 ). digestion was performed with collagenase ( 1 mg / ml ) in a 25 ml flexible plastic bottle under an atmosphere of carbogen ( 95 % o 2 , 5 % co 2 ) for 40 min at 37 ° c . with vigorous shaking . five milliliters of buffer was then added , and the cells were passed through a mesh screen . the cells were then allowed to stand for several minutes in a 15 ml plastic test tube at room temperature , floating , and the buffer underneath was removed . this procedure ( suspension , floating , and removal of buffer underneath ) was repeated three times . adipocyte suspensions ( 3 × 10 5 cells / ml ) were divided into plastic vials ( 0 . 5 ml per vial ) and incubated for 60 min at 37 ° c . under an atmosphere of carbogen with 0 . 2 mm [ u - 14 c ] glucose , in either the absence or presence of insulin . lipogenesis was terminated by adding toluene - based scintillation fluid ( 1 . 0 ml per vial ) and the radioactivity in extracted lipids was counted ( moody et al ., 1974 ). in a typical experiment insulin - stimulated lipogenesis was 4 - 5 fold higher than basal ( basal “ 2000 cpm per 3 × 10 5 cell / h ; v insulin 8 , 000 - 10 , 000 cpm per 3 × 10 5 cells / h ). in this assay insulin stimulates lipogenesis with ed 50 value = 0 . 15 ± 0 . 03 ng / ml ( shechter and ron , 1986 ). an insulin analog exhibiting ed 50 value = 15 ng / ml is considered to have ˜ 1 % of the biological potency of the native hormone . in this assay insulin activates its own receptor to phosphorylate a random copolymer containing l - glutamic acid and l - tyrosine at 4 : 1 molar ratio [ poly ( glu 4 tyr )] seq id no : 1 . the standard enzyme assay mixture ( final volume 60 μl in 50 mm hepes , ph 7 . 4 - 0 . 1 % triton x - 100 ) contained wga purified insulin receptor ( 5μg protein ), 20 mm mgcl 2 , 2 mm mncl 2 , 100 μm atp and varying concentrations ( from 1 ng / ml to 10 mg / ml ) of insulin or insulin derivative . following a 30 min preincubation at 22 ° c ., the reaction was initiated by adding poly ( glu 4 tyr ) seq id no : 1 ( final concentration 0 . 7 mg / ml ), it proceeded for 20 min at 22 ° c . and was terminated by adding edta ( 20 mm ). phosphotyrosine content in polyglu 4 tyr was quantitated by a radio - immunoassay procedure using specific monoclonal antibodies to phosphotyrosine ( final dilution 1 : 100 , 000 ) and 125 i - bsa - phosphotyrosine conjugate . in this particular assay insulin facilitates half maximal effect at a concentration of 20 ± 3 ng / ml . an insulin analog exhibiting ed 50 value = 2 mg / ml is considered as having ˜ 1 % the biological potency of the native hormone . human insulin ( 100 mg , 17 . 2 μmoles ) ( kindly donated by biotechnology - general , rehovot , israel ) was suspended in 4 ml of analytical grade dimethylformamide ( dmf ) containing 17 . 4 mg ( 172 μmoles ) triethylamine . fmoc - osu ( 58 mg , 172 μmoles ) was then added . the homogeneous reaction mixture was stirred at 25 ° c . for 20 hours and ethylacetate was then added until the solution became turbid , followed by the addition of ether to complete precipitation . the solvent was removed by centrifugation and the precipitate washed twice with ether and twice with water . this procedure yielded a mixture of mono , di and tri - n - fmoc - insulin derivatives which were separated and purified by preparative hplc . the monomodified n - fmoc - insulin derivatives can be separated from the di and tri modified derivatives by washing the crude solid with isopropanol . the crude solid was subjected to reversed - phase hplc ( spectra - physics sp 8800 liquid chromatography system ) equipped with hplc prepacked column ( merck , lichroscart 250 - 10 mm containing lichrosorb rp - 18 [ 7 μm ]. a linear gradient was formed from 0 . 1 % trifluoroacetic acid ( tfa ) in h 2 o ( solution a ) and 0 . 1 % tfa in acetonitrile : h 2 o , 75 : 25 ( solution b ). the flow rate was 1 ml / min . the n -( fmoc ) 1 - insulin derivatives emerged from the column with retention times of 21 . 1 , 21 . 9 and 22 . 8 min , the n -( fmoc ) 2 - insulin derivatives with retention times of 26 . 3 , 27 . 1 and 27 . 7 min , and the n -( fmoc ) 3 - insulin with retention time of 31 . 5 min . the fractions corresponding to 21 - 23 min , 26 - 28 min and 31 . 5 min were pooled , lyophilized and chemically characterized . the fractions corresponding to 21 - 23 min ( monomodified insulins ) and to 26 - 28 min ( dimodified insulins ) were further purified to the individual mono - fmoc and di - fmoc - insulin derivatives , respectively . amounts of fmoc groups attached to the insulin molecule were determined spectrophotometrically at 301 nm , following treatment of known amounts of n - fmoc - insulin derivatives with 50 % piperidine in ch 2 cl 2 . following preparative separation by hplc procedure , seven n - fmoc - insulin derivatives were obtained . this includes three monomodified , three dimodified and one trimodified derivative , respectively ( table i ). the retention times and the yields of each individual compound is given in table i . the different n - fmoc - insulins can be readily resolved from each other and obtained in purified form under the experimental conditions applied here . phe b1 , lys b29 - n -( fmoc ) 2 - insulin ( retention time = 27 . 7 min ) and gly a1 , phe b1 , lys b29 - n -( fmoc ) 3 - insulin were characterized by several procedures including mass spectra ( table i ). ( d ) synthesis of phe b1 , lys b29 - n -( fmoc ) 2 - insulin , since the bioassays revealed that phe b1 , lys b29 - n -( fmoc ) 2 - insulin is especially suited as a long - acting insulin , an alternative procedure was designed for synthesizing it . the gly a1 moiety was specifically protected with the t - boc group under designed experimental conditions , using one equiv of di - tert - butyldicarbonate and dmso / et 3 n , 20 : 1 , as a solvent . following separation by hplc procedure , gly a1 - n - boc - insulin was reacted with excess of fmoc - osu ( 10 equiv ), using dmf as a solvent and diea as a base . treatment with tfa and purification on hplc produced phe b1 , lys b29 - n -( fmoc ) 2 - insulin , in a good yield (˜ 50 %). several features of the n - fmoc - insulin derivatives of the present invention are summarized in tables ii and iii . solubility of the derivatives in aqueous solutions decreased with increased modification . n -( fmoc ) 1 - insulin are only slightly less soluble than native insulin whereas n -( fmoc ) 3 - insulin is about 20 fold less soluble than the native hormone . the biological potencies decreased as well with increased derivatization . thus , mono , di and tri - n - fmoc - insulins exhibit 40 - 80 %, 2 - 9 % and & lt ; 1 % of the native insulin biological potency , respectively , as judged by [ poly ( glu 4 tyr )] seq id no : 1 phosphorylating assay . according to the more sensitive biological assay of lipogenesis with intact rat adipocytes , the fmoc - insulin derivatives exhibit much lower biological potencies . thus , gly a1 - n - fmoc - insulin and di - n - fmoc - insulins exhibit 4 . 7 % and 0 . 4 - 1 . 4 % of the native insulin biological potency , respectively , using the lipogenesis assay ( table iii ). all seven derivatives are reverted into the native hormone upon incubation for 2 days at ph 8 . 5 . this was proved both by regaining the full biological potency ( tables ii and iii ) and by disappearance of the peaks of the derivatives , along with the appearance of the native hormone peak ( retention time = 15 min ) upon separation by analytical hplc procedure . amino acid analysis was used to verify the correct composition of all the fmoc - derivatives . a established with merck , lichrospher 100 rp - 8 ( 5 μm ) column , employing a linear gradient formed from 60 % solution a ( 0 . 1 % tfa in water ) and 40 % solution b ( 0 . 1 % tfa in acetonitrile : water , 75 : 25 ), to 100 % solution b in 40 minutes ( flow rate of 1 ml / min ). insulin - like potency was determined by both biological assays described in the experimental part . prior to studying the antidiabetic potencies of n - fmoc - insulins in diabetic rats , their rate of reactivation ( which represents their conversion to the native hormone ) was tested in the test tube . the derivatives have been dissolved in hepes - buffer ( 50 mm , ph 7 . 4 ) containing 10 % dimethylsulfoxide ( dmso ) and incubated at 37 ° c . ( i . e . at physiological ph and body temperature ). at the indicated time points aliquots were withdrawn for determining the biological potency relative to native insulin . biological activities were evaluated by insulin receptor tyrosine kinase activation ( a cell free assay as described in biological procedures above , section ( iv )) and by stimulation of lipogenesis in intact rat adipocytes as described in biological procedures , section ( ii ). the results are shown in fig1 . half maximal activation occurred for n -( fmoc ) 3 - insulin at t ½ = 14 days and nearly full activation was evident following 21 days of incubation . as a rule , the activation rate of n -( fmoc ) 2 - insulin at ph 7 . 4 was faster . the six day lag period observed with n -( fmoc ) 3 - insulin was not seen with n -( fmoc ) 2 - insulin . thus n -( fmoc ) 3 - insulin appears to have a slower hydrolyzable fmoc moiety that limits reactivation of the analog . following its hydrolysis , the rate of activation is increased . from a practical viewpoint it implies that the mixture of the two analogs may release active insulin over a prolonged period of time , by covering earlier and later time periods . the relatively active monomodified fmoc - insulin regained full biological potency at ph 7 . 4 with t ½ = 5 days . once arriving at the circulation n -( fmoc ) 2 - and n -( fmoc ) 3 - insulins are expected to provide a low basal concentration of the hormone over prolonged periods . this may be primarily dictated by the rate of conversion of the inactive derivative into the active short - lived native hormone . fortunately , the n -( fmoc ) 2 , 3 - insulins exhibit slow rates of activation ( as shown in fig1 ). a rapid ( sudden ) rate ( i . e . within minutes rather than hours ) could have been resulted in hypoglycemic episodes phe b1 , lys b29 - n -( fmoc ) 2 insulin is 9 % biologically active , as determined by the [ poly ( glu 4 tyr )] seq id no : 1 phosphorylating assay , and exhibits intermediate solubility between the native and n -( fmoc ) 3 - insulin . unlike n -( fmoc ) 3 - insulin , the conversion of ( n - fmoc ) 2 - insulin to the native hormone starts shortly ( within hours ) after incubation , as shown in fig1 . thus , n -( fmoc ) 2 - insulin ( being a priori more active ) is expected to have a faster onset of action following administration . thus , a proper combination of n -( fmoc ) 2 - and ( n - fmoc ) 3 - insulins can make the ideal formula for basal insulin release that is characterized by rapid onset and long duration of action , following a single administration , and constitutes one preferred embodiment of the invention . effect of a single administration of phe b1 , lys b29 - n -( fmoc ) 2 - ( b ) effect of a single intraperitoneal administration of phe b1 , lys b29 - n -( fmoc ) 2 - insulin to normal rats in order to further confirm that the long - acting capacity of ( fmoc ) 2 - insulin is due to its slow conversion to the native hormone under physiological conditions , an additional in vivo assay has been designed . this assay reflects the post - subcutaneous long - acting feature of insulin ( or insulin derivatives ) within the circulation . normal rats received a single intraperitoneal injection of native insulin , nph - insulin and ( fmoc ) 2 - insulin ( 3 mg / rat ), and their blood glucose levels were monitored over a period of three days . the results are shown in fig2 . thus , native insulin ( fig3 ) and nph - insulin ( fig2 ) induced hypoglycemia over a period of about 12 and 15 hours , respectively . recovery from hypoglycemia occurred with t ½ of 8 and 10 hours , respectively . ( fmoc ) 2 - insulin reduced blood glucose levels over a period of about 48 hours , and recovery from hypoglycemia occurred with t ½ of 26 hours . these results supported our notion that indeed the prolonged action of ( fmoc ) 2 - insulin is emerging from its intrinsic characteristics , and not due to the conventional mechanism of nph - insulin , i . e . precipitation at the injection site and its slow dissolution - entrance to the circulation . as expected , the differences in the capacities of native insulin and nph - insulin to reduce blood glucose levels in this assay are minor as compared to their administration by the subcutaneous mode . this fact is due to the large volume of liquids existing in the peritoneal cavity and hence fast conversion of the zn - crystalline insulin to yield the monomeric form . the two other ( fmoc ) 2 - insulin derivatives , [ gly a1 , lys b29 ] and [ gly a1 , phe b1 ], were synthesized and also evaluated in this assay . results ( not shown ) exhibited similar long - acting pattern as for phe b1 , lys b29 - n -( fmoc ) 2 - insulin , with t ½ of 22 - 24 hours for both derivatives . native insulin or n -( fmoc ) 3 - insulin ( 1 mg / ml of each in 50 mnm hepes , ph 7 . 4 , 10 % dmso ) were incubated at 37 ° c . chymotrypsin and trypsin ( 0 . 5 % w / w , of each ) was then added . at the indicated time points aliquots were withdrawn and subjected to analytical hplc procedure . percent degradation was determined by the decrease in area of the native insulin peak ( retention time 15 min ) and of the n -( fmoc ) 3 - insulin peak ( retention time 31 . 5 min ). the results are shown in fig4 . n -( fmoc ) 3 insulin was found to be highly resistant to proteolysis by a mixture of chymotrypsin and trypsin at ph 7 . 4 . proteolysis proceeded with t ½ values of 0 . 5 and 7 . 5 hours for the native and n -( fmoc ) 3 insulin , respectively . effect of a single subcutaneous administration of phe b1 , lys b29 - n -( fmoc ) 2 - and n -( fmoc ) 3 insulin to stz - diabetic rats the stz rat is an excellent model for evaluating in vivo insulin therapy . in this model about 90 % of the β - cell function has been destroyed by streptozotocin , as judged by histological studies ( as described by pederson et al ., 1989 ). the rats are hypoinsulinemic ( having 10 - 30 % of the normal insulin level ), hyperglycemic (& gt ; 300 mg / dl ; normal glucose level of control rats is 90 - 100 mg / dl ), and catabolic . visible external symptoms are ‘ sick ’ appearance and three to four fold increase in fluid intake and urine excretion . daily weight gain is decreased to 10 - 20 % ( 0 . 3 - 0 . 8 g / day / rat ) of the normal weight gain of control rats . the pathological changes in tissues of stz - rats are enormous . some of the more prominent biochemical alterations are decreased activity of key enzymes of glycogen metabolism , depletion in liver glycogen , a decrease in number of glucose transporters , in peripheral tissues , and an increase in insulin binding capacity that is not accompanied with an increase in responsiveness to insulin . in this diabetic rat model a convenient insulin therapy is the continuous administration of insulin ( 5 units / day / rat ) over a period of one week . this treatment normalizes blood glucose levels , returns diabetic rats into anabolic state , and ameliorates many of the pathological effects induced by hyperglycemia and hypoinsulinemia . a single administration of rapid - acting ( regular ) insulin is effective over a period of several hours only . also , following termination of the seven day therapy protocol , hyperglycemia reoccurs within 24 - 30 hours . to evaluate whether n -( fmoc ) 3 - insulin has prolonged antidiabetic effect , stz - rats , two weeks after induction of diabetes , were administered either a single subcutaneous injection of native insulin ( group a , 25 units , 1 mg , dissolved in 1 . 0 ml h 2 o - 10 % dmso , n = 4 ), or of n -( fmoc ) 3 - insulin ( group b , 1 mg , dissolved in 1 . 0 ml h 2 o - 10 % dmso , n = 4 ). blood glucose levels and daily weight gains were followed over a period of seven days . the results are shown in fig5 . each point represents the arithmetic mean ± sem of plasma glucose for 4 rats . circulating glucose levels in group b were significantly lower . thus , glucose levels were 90 - 110 mg / dl lower in group b , starting from day two following administration , and these lower glucose levels existed up to day six . on day seven , there was no significant difference between the two groups of rats in circulating glucose levels . the rats receiving n -( fmoc ) 3 insulin had a ‘ healthier ’ appearance . daily weight gains were nearly three times higher in group b and amounted to 0 . 57 ± 0 . 08 and 1 . 43 ± 0 . 14 g / rat / day , in groups a and b , respectively ( not shown ). thus , a single administration of n -( fmoc ) 3 - insulin gave prolonged and satisfactory antidiabetic actions lasting over a period of four days ( following a delayed onset of approximately two days ). this long - lasting effect in vivo may be explained by the ability of the derivative to escape receptor - mediated endocytosis and also by its resistance to proteolysis . in addition n -( fmoc ) 3 insulin is largely insoluble in aqueous solutions . thus , in humans , the overall lasting effect may proceed by substitution of the old therapeutic principle , i . e . gradual dissolution of ‘ built - in ’ insoluble insulin , following subcutaneous administration together with the new principle , namely , circulating long - living covalently modified inactive insulin derivative , that is slowly converted into the native hormone . as this insulin derivative is inactive , larger doses can be administered with no fear of hypoglycemic episodes . to test the effect of phe b1 , lys b b29 - n -( fmoc ) 2 - insulin in lowering blood glucose levels in experimental diabetic rats , stz - treated rats , 9 days after induction of diabetes , received either a single s . c . injection of the n -( fmoc ) 2 - insulin ( group a , 3 mg / rat , in 2 . 0 ml 20 % dmso , n = 5 ), or a single s . c . injection of long - acting insulin ( nph - human insulin , humulin n , hi - 310 ) ( group b , 0 . 75 ml ( 3 mg ) per rat , n = 5 ). group c ( n = 5 ) received the vehicle only ( 2 . 0 ml of 20 % dmso ). blood glucose levels were determined daily . the results are shown in fig6 in which the horizontal dashed line indicates the arithmetic mean of plasma glucose for control rats . as shown in fig6 a single subcutaneous administration of hplc - purified phe b1 , lys b b29 - n -( fmoc ) 2 - insulin induced normoglycemia over a period of 4 days and increased daily weight gain of these catabolic stz - rat models ( table iii ). n -( fmoc ) 2 - insulin was as effective as the commercially available ( insoluble ) long - acting preparation . rapid - acting ( soluble ) insulin is effective in lowering blood glucose levels in this experimental system over a period of several hours only ( not shown ). this preparation is fairly soluble in aqueous solution ( at ph 7 . 4 ), a fact that may be advantageous over the insoluble n -( fmoc ) 3 - insulin , because suspensions cannot be accurately administered by s . c . injection . the fmoc group itself was modified in order to reduce fmoc - insulin &# 39 ; s hydrophobicity and consequently to increase its solubility in aqueous buffers , as well as to modify the rate of reconversion to insulin . this can be done by introducing polar or , preferably , charged groups into the fluorene ring , such as halogen , nitro , carboxyl , amino , ammonium , and sulfo groups . in electrophilic substitution reactions , fluorene is first attacked at the 2 position and , generally , the nature of the substituent at position 9 ( e . g . ch 2 oco - osu ) has no effect on the orientation of the substitution . thus , treatment of fmoc - osu with 0 . 9 equiv of chlorosulfonic acid in dichloromethane ( dcm ) at 0 ° c ., gave ( 2 - sulfo ) fmoc - osu in high yield ( formula ( i ), r 1 = so 3 h at position 2 , r 2 = r 3 = r 4 = h , a = oco - osu ). treatment with more than 1 equiv will result in substitution also at position 7 of the fluorene ring . the ( 2 - sulfo ) fmoc groups were introduced into insulin by coupling of the active ( 2 - sulfo ) fmoc - osu ester to the amino groups of insulin . reaction was carried out in aqueous buffers ( ph 7 . 4 ) and excess reagent (˜ 20 equiv ). following dialysis and lyophilization the product was turned to be water soluble . hplc analysis showed predominance of one main product , apparently ( sulfmoc ) 2 - insulin , as determined by mass - spectra ( m / z 6411 ). when the reaction was carried out in acetonitrile / water , 1 : 1 , the main product was ( sulfmoc ) 3 - insulin , as determined by mass - spectra ( m / z 6713 ). the ( sulfmoc ) 2 - insulin was fully reverted into the native hormone upon incubation at 37 ° c . for 36 hours at ph 8 . 5 ( 0 . 1 m nahco 3 ), or for 10 days at ph 7 . 4 ( 50 mm hepes buffer ) with t ½ values of 12 - 15 hours and 6 days , respectively . this was proved by disappearance of the peak of the insulin derivative , along with the appearance of the peak of the native hormone , using analytical hplc procedure . note that hydrolysis of ( sulfmoc ) 2 - insulin to the native hormone is faster as compared to the hydrolysis of ( fmoc ) 2 - insulin ( 21 days at ph 7 . 4 , 37 ° c .). ( sulfmoc ) 2 - insulin is 0 . 5 % biologically active and exhibits good solubility in water . upon incubation at ph 8 . 5 , 37 ° c ., ( sulfmoc ) 2 - insulin returns to fully - active native insulin with t ½ value of 4 - 6 hours as judged by time - dependent increase in biological potency ( assay of lipogenesis in rat adipocytes ). ( c ) effect of a single intraperitoneal administration of ( 2 - sulfo ) fmoc - insulin to normal rats to evaluate the long - acting capacity of ( sulfmoc ) 2 - insulin , downstream to subcutaneous absorption , normal rats were received a single intraperitoneal injection of native insulin , nph - insulin , or ( sulfmoc ) 2 - insulin . blood glucose levels were followed for two days . fig3 shows that ( sulfmoc ) 2 - insulin induced hypoglycemia over a period of 24 hours . recovery from hypoglycemia occurred with t ½ value = 14 h . in case of rapid and nph - insulin , these t ½ values amounted to 8 ( fig3 ) and 10 hours ( fig2 ), respectively . thus , a single administration of ( sulfmoc ) 2 - insulin gave an intermediate anti - diabetic action , that is 1 . 5 - 2 fold longer than that of rapid or nph - insulin . these results agree with the accelerated rate of hydrolysis of sulfmoc - insulin previously found in vitro . thus , the sulfonic acid group at position 2 of the fluorene ring increased the rate of proton abstraction at position 9 and therefore the hydrolysis of the sulfmoc moiety , by 2 - 3 fold . earlier studies performed on the lability of the sulfmoc group to various bases , showed greater base sensitivity than the parent system . in addition , rate constant for the release of the sulfmoc group from glycine , for example , was much higher than the fmoc group ( factor of about 30 ). therefore , increase of solubility by introducing the sulfmoc group into insulin , reciprocally decrease the long - lasting effect of this derivative . thus , the principle of long - lasting anti - diabetic effect , through escaping receptor - mediated endocytosis and degradation , holds for the sulfonic - insulin derivative as well . the enhanced rate of the removal of the sulfmoc group from insulin may be advantageous in certain applications of insulin administration such as intermediate preparations . such preparations will obviously have the benefit of being completely soluble in aqueous buffers , in contrast to the available commercial preparations . moreover , other groups , with diverse acidities or polarities can be introduced to the fluorene ring . therefore , controlling of the type and number of groups introduced to the fmoc group , can determine the degree of solubility and the rate of reactivation of the modified insulin . n -( fmoc ) 3 - insulin ( 64 . 5 mg ; 10 μmol ; 60 μmol of carboxylic moieties , i . e . pertaining to 4 intrachain glu and two c - terminal residues ) was dissolved in 8 ml of dimethylformamide ( dmf ) ( lab . scan ., dublin , ireland ) along with o - nitrophenol ( 250 μmol ; 35 mg ) or n - hydroxysuccinimide ( 250 μmol ; 28 mg ) and the solution was cooled to 4 ° c . a solution of n , n - dicyclohexylcarbodiimide ( dcc ; 250 mmol , 53 mg ) in 0 . 5 ml of dmf was added and the reaction mixture was kept 1 h at 4 ° c . and then 6 h at room temperature . precipitated n , n - dicyclohexyl urea was removed by centrifugation and the o - nitrophenyl or n - hydroxysuccinimide esters , respectively , of n -( fmoc ) 3 - insulin were precipitated by dry , ice cold , ether . the solid was washed twice with dry ether , dried and dissolved in 8 ml of dmf . a a solution of 9 - fluorenylmethanol ( 250 μmol ; 50 mg ) and imidazole ( 250 μmol ; 17 mg ) in 1 ml dmf was added . the reaction mixture was allowed to stand overnight at room temperature . precipitation with dry ether afforded 62 mg of n -( fmoc ) 3 , c -( fm ) n - insulin . the main reaction product was the hexa - 9 - fluorenylmethyl ester c -( fm ) 6 of n -( fmoc ) 3 - insulin ( n = 6 ). for the preparation of t - butyloxycarbonyl ( t - boc ) 3 - insulin , di - tert - butyldicarbonate ( 56 mg , 258 μmole ) was added to an ice - cooled , stirred suspension of insulin ( 100 mg , 17 . 2 μmole ) and triethylamine ( 174 mg , 172 μmole ) in dmf ( 4 ml ), the reaction mixture was allowed to warm to room temperature and stirred for 5 h ( gradually becoming clear ). ethyl acetate was then added until the solution became turbid , followed by addition of ether , and the precipitate was centrifuged and washed twice with ether . the resulting crude solid ( 95 mg ) was utilized further without any purification . analytical hplc showed one main product that eluted at 27 . 5 min . the product was dissolved in 10 ml of dmf and treated with o - nitrophenol or n hydroxysuccinimide , as above , to yield the corresponding active esters . these were reacted with 9 - fluorenylmethanol , in the presence of imidazole , as above , and n -( t - boc ) 3 , c -( fm ) n - insulin was obtained as a powder ( 87 mg ) after precipitation with dry ether . the powder was dried in vacuum over p 2 o 5 and then treated for 1 h at room temperature with 5 ml of trifluoroacetic acid to effect removal of n - terminal t - boc protecting groups . during this procedure , most of the insulin derivative dissolved . addition of ice - cold dry ether led to a powder which was isolated by centrifugation and washed thoroughly with dried ether . yield of a product , primarily c -( fm ) 6 - insulin , was 79 mg . under normal physiological conditions , hgh levels in healthy subjects are increased daily in a pulsatile manner several times ( at day and night ). hgh is a short - lived species . the current therapeutic protocol includes a single daily injection of hgh and is likely to be effective for several hours only . a long - acting ( slow - released ) hgh preparation that supply a treshold - based level of hgh during 24 h hours of the day and night , is highly desirable . native hgh ( biotechnology general , rehovot , israel ; 9 . 2 mg ) was dissolved in 0 . 1 m nahco 3 ( 2 . 0 ml ; ph 8 . 5 ), dmso ( 0 . 1 ml ) was added ( final dmso concentration . ˜ 5 %), and the solution was cooled to 0 ° c . one equivalent of fmoc - osu in 10 μl ( taken from a stock solution of 18 . 5 mg / ml in dmso ) was then added , and the reaction proceeded for 30 min at 0 ° c ., under moderate stirring . another 10 μl ( containing 1 equivalent of fmoc - osu ) was then added , and after 30 min the mixture was dialyzed overnight at 7 ° c ., against h 2 o . semi - preparative hplc yielded two main protein peaks corresponding to native hgh (˜ 20 % of total ; retention time 30 min ; rp - 8 column ; 250 × 10 mm ; merck ) and modified fmoc - hgh (˜ 80 % of total ; retention - time 32 min ). table v shows conversion of fmoc - hgh ( 1 mg / ml ) to native hgh upon incubation at 37 ° c . in 0 . 1 m nahco 3 ( ph 8 . 5 ). at the indicated time points , aliquots were withdrawn and subjected to analytical hplc procedure ( rp - 8 column ). conversion of fmoc - hgh to native hgh was evaluated by the increase in peak area corresponding to native hgh . as shown in table v , fmoc - hgh has 15 % of the native hormone &# 39 ; s receptor - binding potency . incubation of fmoc - hgh at ph 8 . 5 ( 37 ° c .) for about 6 days or at ph 10 . 5 ( 37 ° c .) for four days yielded fully active , native hgh , as judged by hplc analysis and by receptor binding assays . generation of native hgh from fmoc - hgh upon incubation at 37 ° c . ( 1 ) displacement of iodinated hgh was carried out according to gertler et al ., 1984 . native hgh was fully stable under the conditions applied to fmoc - hgh ( ph 10 . 5 , 37 ° c ., 4 days ). ( 2 ) fmoc - hgh ( 1 mg / ml ) was incubated at 37 ° c . in 0 . 1 m nahco 3 ( ph 8 . 5 ). at the indicated time points , aliquots were withdrawn and subjected to analytical hplc . conversion of fmoc - hgh to native hgh was evaluated by the increase in peak area corresponding to native hgh . cephalexin [ 7 -( d - α - aminophenylacetamido ) desacetoxycephalosporanic acid ) is a β - lactam antibiotic with a broad spectrum of activity against gram (−) and gram (+) bacteria . two monosubstituted cephalexins were prepared by covalently attaching a fluorenylmethyl ( fm ) moiety either to the amino group ( n - fmoc - cephalexin ) or to the carboxylic group via esterification ( cephalexin - o - fm ). to a stirred suspension of cephalexin hydrate ( sigma , usa ; 50 mg , 0 . 144 mmole ) and triethylamine ( 29 mg , 0 . 288 mmole ) in dichloromethane ( dcm ; 2 . 5 ml ), a solution of fmoc - osu ( 145 mg , 0 . 432 mmole ) in dcm ( 2 . 5 ml ) was added dropwise over 5 min . the reaction mixture , which became clear after 1 h , was stirred overnight at room temperature , during which it turned turbid . after concentration under vacuum , ether was added to the mixture , and the resulting precipitate was filtered and washed twice with ether . the filtrate was dissolved in dcm and extracted with acidified water ( ph ˜ 2 ), water and brine , and dried over anhydrous mgso 4 . after filtration and concentration of the solution , ether was added . the precipitated product was filtered and washed with ether to yield fmoc - cephalexin as a pure product , as judged by tlc ( 1 - butanol : acetic acid : water , 8 : 1 : 1 ) and by analytical hplc procedure ( eluted at 33 min , while under similar conditions native cephalexin is eluted at 6 . 5 min ). mass spectrum analysis ( fast atom bombardement , fab ) gave the expected molecular weight for n - fmoc - cephalexin ( m / z 570 . 1 [ m + h ] + ). for the determination of the antibacterial potencies of native cephalexin and fmoc - cephalexin , tubes containing a dilute suspension of staphylococcus aureus ( 0 . 5 ml / glass tube ) were incubated for 6 hours at 37 ° c . in the absence and presence of increased concentrations of native cephalexin or fmoc - cephalexin , prior to and subsequent to the treatment specified in air table vi . at the indicated time points , aliquots were withdrawn and analyzed for their potency to arrest growth of staphylococcus aureus . bacterial growth was then evaluated by increased turbidity measured spectroscopically at 700 nm . ( 1 ) numbers refer to the potency of cephalexin employed as a control throughout the assays . incubations were performed at ph 7 . 4 with native cephalexin or with n - fmoc - cephalexin ( 100 μg / ml ) in 50 mm hepes buffer , 20 % dmso . as shown in table vi , n - fmoc - cephalexin is inactive (˜ 6 %). preincubation over a period of 6 days ( ph 7 . 4 , 37 ° c .) generated native cephalexin as judged by hplc monitoring , and by regaining about 50 % of the antibacterial potency of the parent compound . native cephalexin undergoes substantial time - dependent spontaneous inactivation upon incubation , whereas n - fmoc - cephalexin is significantly more stable to the same conditions . thus , the expected prolonged action of n - fmoc - cephalexin in vivo is likely to originate both from higher chemical stability at physiological ph and temperature , as well as by escaping degradation . n - fmoc - cephalexin is more stable to hydrolysis by penicillinase than the parent compound ( not shown ). to an ice - cooled stirred suspension of cephalexin hydrate ( 50 mg , 0 . 144 mmole ) and triethylamine ( 29 mg , 0 . 288 mmole ) in dcm ( 3 ml ), a solution of di - tert - butyl dicarbonate ( 94 . 2 mg , 0 . 432 mmole ) in dcm ( 2 ml ) was added . the reaction mixture was allowed to warm up to room temperature and stirred overnight , until no ninhydrin - positive starting material was observed by tlc ( 1 - butanol : acetic acid : h 2 o , 8 : 1 : 1 ). the reaction mixture was then diluted with dcm ( 2 . 5 ml ), extracted with acidified water ( ph ˜ 2 ), water and brine , and dried over anhydrous mgso 4 . after concentration , the product was precipitated with petroleum ether ( b . p . 40 - 60 ° c . ), filtered and washed again with petroleum ether . the product was homogeneous as confirmed by tlc and hplc procedures . to an ice - cooled stirred solution of n - boc - cephalexin ( 25 mg , 0 . 056 mmole ), 9 - fluorenylmethanol ( 22 mg , 0 . 112 mmole ) and 4 - dimethylaminopyridine ( 13 . 7 mg , 0 . 112 mmole ) in dcm ( 2 ml ), a solution of dcc ( 23 . 1 mg , 0 . 112 mmole ) in dcm ( 1 ml ) was added dropwise over 20 min . the resulting mixture was stirred overnight at room temperature and the solution was filtered to remove dicyclohexylurea . the filtrate was diluted with dcm ( 2 . 5 ml ) and then extracted twice with 1 m nahco 3 solution , 10 % citric acid , water and brine , and dried over anhydrous mgso 4 . the solution was evaporated to yield an oily solid , which was triturated with petroleum ether to furnish the boc - cephalexin - ofm product ( as confirmed by tlc and hplc ). the boc moiety was removed by dissolving the crude solid ( 25 mg ) in 1 ml solution of tfa : dcm ( 1 : 1 v / v ). after standing for 20 min at room temperature , the tfa was removed by evaporation and the solid residue dissolved in isopropanol which was then evaporated . this procedure was repeated twice . the crude solid was triturated with petroleum ether to form the final pure ester , as judged by both tlc and hplc procedures . polymyxin - b ( pmxb ), a representative member of the family of cyclic - peptidic antibiotics , is effective against gram (−) bacteria . pmxb contains 5 residues of diaminobutyric acid which can be modified by insertion of fmoc groups using the reagent 4 -( 9 - fluorenylmethoxycarbonyloxyl ) phenyl - dimethylsulfonium methylsulfate ( fmoc - dsp ). the molar ratio of fmoc - dsp reagent and peptide will determine the extent of modification of the pmxb molecule . for the preparation of ( fmoc ) 2 - pmxb , to a stirred solution of pmxb ( sigma , usa : 10 mg , 7 . 2 μmole ) and ( fmoc - dsp ; 7 . 1 mg , 14 . 4 μmole ) in h 2 o ( 1 ml ), a solution of nahco 3 ( 0 . 1 m , 0 . 15 ml ) was added dropwise . the reaction mixture which gradually became turbid was stirred overnight , at room temperature . the resulting precipitate was centrifuged , washed twice with water , dissolved in a small volume of dmf and precipitated with ether to give white crude solid . ( 1 ) numbers refer to the potency of pmxb employed as a control throughout the assays . ( 2 ) incubation was carried out at ph 8 . 5 with pmxb , or ( fmoc ) 2 - pmxb ( 100 μg / ml ) in 0 . 1 m nahco 3 containing 1 % dmso . ( 3 ) incubation was carried out at ph 7 . 4 with pmxb , or ( fmoc ) 2 - pmxb ( 100 μg / ml ) in 50 mm hepes - buffer ( ph 7 . 4 ), containing 1 % dmso . analytical hplc ( rp - 18 ; 250 × 4 mm ; merck ) employing linear gradient formed from 70 % solution a ( 0 . 1 % tfa in water ) and 30 % solution b ( 0 . 1 % tfa in acetonitrile : water , 75 : 25 ), to 100 % solution b in 40 minutes ( flow rate of 0 . 8 ml / min ), showed one main product eluted at 39 min ( retention time of pmxb under these conditions is 13 . 5 min ). the crude solid was applied to preparative hplc to provide a pure product . analysis of ( fmoc ) 2 - pmxb by mass spectrum ( fab ), gave the expected molecular weight for this compound ( m / z 1647 [ m - h ] + ). the antibacterial potencies of ( fmoc ) 2 - pmxb and of native pmxb were assayed under various experimental conditions as follows : a dilute suspension of e . coli ( 0 . 5 ml per glass test tube ) was incubated for 6 hours at 37 ° c . without or with increased concentrations of ( fmoc ) 2 - pmxb and native pmxb . at the indicated time points aliquots were withdrawn and analyzed for their potencies to arrest growth of e . coli , and bacterial growth was then evaluated by measuring turbidity at 700 nm . as shown in table vii , ( fmoc ) 2 - pmxb is inactive (˜ 1 %) and is largely hydrolyzed back to active pmxb with t ½ values of ˜ 3 and ˜ 1 days at ph 8 . 5 and 7 . 4 , respectively . piperacillin ( 4 - ethyl - 2 , 3 - dioxopiperazinecarbonyl ampicillin ) is a broad spectrum semi - synthetic antibiotic related to penicillin , which is not effective when administered orally . piperacillin ( free carboxyl ) was prepared from piperacillin sodium salt ( sigma , usa ) by acidic extraction with ethyl acetate . piperacillin - ofm was synthesized as described for cephalexin - ofm ester in example 8 above , by reacting 1 equivalent of carboxyl with 2 equivalents , each , of 9 - fluorenylmethanol , 4 - dimethylaminopyridine and dcc . the crude solid was recrystallized from dcm - ether , yielding a pure product as confirmed by tlc and hplc procedures . propananolol [ 1 -( isopropylamino )- 3 -( 1 - naphthyloxy )- 2 - propanol ], a representative member of the beta blockers family , is a β - adrenergic antagonist used as antihypertensive , antianginal and antiarrhythmic . propranolol binds , but does not activate , the β - adrenergic receptor . competition for these sites with β - adrenergic antagonists reduces pathological hypertensive states . patients receive propranolol orally on a daily basis . however , a vast majority of β - adrenergic antagonists are rather hydrophilic in nature , and are not absorbed efficiently while administered orally , e . g . acetylbutolol , athenolol , betaxolol , carteolol , nadolol , and sotalol . for the preparation of fmoc - propranolol , a solution of fmoc - osu ( 170 mg , 0 . 50 indole ) in dcm ( 2 . 5 ml ) was added dropwise over 5 min to a stirred solution of (±)- propranolol hydrochloride ( 50 mg , 0 . 17 mmole ) and triethylamine ( 34 mg , 0 . 34 mmole ) in dichloromethane ( dcm , 2 . 5 ml ). after stirring overnight at room temperature , the reaction mixture was extracted with acidified water ( ph ˜ 2 ), water and brine , and dried over anhydrous mgso 4 . the solution was evaporated to furnish crude solid which was then triturated with hexane to give fmoc - propranolol . the product was examined by tlc ( 1 - butanol : acetic acid : water , 8 : 1 : 1 ) and hplc , and proved to be pure ( retention times of propranolol and fmoc - propranolol , under the same conditions , are 16 and 51 min , respectively ). mass spectrum ( fab ) gave the correct m / z : 482 . 2 [ m + h ]+ + . the β - adrenergic potency of fmoc - propranolol was analyzed . the results are shown in fig7 . freshly prepared rat adipocytes were incubated for 2 hrs at 37 ° c ., with isoproterenol ( final concentration 1 μg / ml , 4 μm ) and the indicated concentrations of propranolol ( circles ), fmoc - propranolol ( filled circles ) or fmoc - propranolol that was incubated for 7 days at 37 ° c ., ph 8 . 5 ( squares ). the amount of glycerol released to the medium was then determined according to shechter , 1982 . ic 50 is the amount of propranolol or n - fmoc - propranolol derivative ( in μm ) that inhibited isoproterenol - mediated glycerol release , half maximally . as shown in fig7 fmoc - propranolol has ˜ 7 % of the potency of native propranolol . incubation of fmoc - propranolol for 7 days at ph 8 . 5 ( 37 ° c .) generated 50 - 70 % of the native drug β - adrenergic potency . the derivative is substantially hydrophobic , a feature that may assist in gastrointestinal absorption . 1 . bodanszky , m . and bednarek , m . 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( 1974 ) horm . metab . res . 6 , 12 - 16 . 9 . pederson , r . a ., ramanadham , s ., buchan , a . m . j . and mcneill , j . h . ( 1989 ) diabetes 38 , 1390 - 1395 . 12 . shechter , y . and ron , a . ( 1986 ) j . biol . chem . 261 , 14945 - 14950