6450
`
`J. Med. Chem. 2007, 50, 6450–6453
`
`8-(3-(R)-Aminopiperidin-1-yl)-7-but-2-ynyl-3-
`methyl-1-(4-methyl-quinazolin-2-ylmethyl)-
`3,7-dihydropurine-2,6-dione (BI 1356), a
`Highly Potent, Selective, Long-Acting, and
`Orally Bioavailable DPP-4 Inhibitor for the
`Treatment of Type 2 Diabetes†
`
`Matthias Eckhardt,*,# Elke Langkopf,*,# Michael Mark,§
`Moh Tadayyon,§ Leo Thomas,§ Herbert Nar,|
`Waldemar Pfrengle,¶ Brian Guth,‡ Ralf Lotz,‡ Peter Sieger,‡
`Holger Fuchs,⊥ and Frank Himmelsbach*,#
`
`Boehringer Ingelheim Pharma GmbH & Co. KG,
`88400 Biberach, Germany
`
`ReceiVed October 11, 2007
`
`Abstract: A new chemical class of potent DPP-4 inhibitors structurally
`derived from the xanthine scaffold for the treatment of type 2 diabetes
`has been discovered and evaluated. Systematic structural variations have
`led to 1 (BI 1356), a highly potent, selective, long-acting, and orally
`active DPP-4 inhibitor that shows considerable blood glucose lowering
`in different animal species. 1 is currently undergoing clinical phase
`IIb trials and holds the potential for once-daily treatment of type 2
`diabetics.
`
`Chart 1
`
`Scheme 1. Syntheses of Compounds 5 and 6 a
`
`a Reagents: (a) PhCH2Cl, Me2CCHCH2Br, or MeCCCH2Br, iPr2NEt,
`DMF, room temp, 4a 75%, 4b 91%, 4c 84%; (b) piperazine (excess), THF
`or MeCN, reflux, 5a 64%, 5b 39%, 5c 98%; (c) 3-aminopiperidine · 2HCl,
`K2CO3, MeCN, 70 °C, 58%.
`
`Type 2 diabetes is establishing itself as an epidemic of the
`21st century with an estimated 5% of the adult world population
`suffering from the disease.1 The number of deaths attributable
`to diabetes is steadily growing, currently estimated at 3.8 million
`cases each year, reflecting the insufficient glycemic control
`achieved with currently available treatments. Therefore, more
`effective therapeutics for glycemic control are badly needed.
`DPP-4a is a protease that specifically cleaves dipeptides from
`proteins and oligopeptides after a penultimate N-terminal proline
`or alanine.2 DPP-4 is involved in the degradation of a number
`of neuropeptides, peptide hormones, and cytokines, including
`the incretins GLP-1 and GIP.3 GLP-1 and GIP are released from
`the gut in response to food intake and exert a potent glucose-
`dependent insulinotropic action and thereby contribute to the
`maintenance of postmeal glycemic control.4 In addition, they
`exhibit beneficial effects on pancreatic (cid:1) cells.5 Other effects
`that have been described for GLP-1 are an inhibition of glucagon
`release from pancreatic R cells, a reduction of food intake, and
`a retardation of gastric emptying.6 Consequently, inhibiting
`DPP-4 prolongs the action of GLP-1 and GIP, which in turn
`
`improves glucose homeostasis with a low risk of hypoglycemia
`and potential for disease modification. Indeed, clinical trials
`involving diabetic patients have shown improved glucose control
`by administering DPP-4 inhibitors,
`thus demonstrating the
`benefit of this promising new class of antidiabetics.7 Intense
`research in the DPP-4 field has resulted in the launch of one
`inhibitor and the advancement of others into preregistration/
`phase III.8
`Herein, we report the discovery of the novel, potent, and
`selective DPP-4 inhibitor 1 (BI 1356)9 originating from the class
`of xanthines (Chart 1).
`Compound 2, discovered through a high-throughput screening
`campaign that involved about 500 000 compounds and showing
`promising inhibitory activity in the low micromolar range, was
`our starting point in the search for an effective DPP-4 inhibitor.10
`Systematic structural modifications on the xanthine scaffold were
`carried out to study the structure–activity relationship and
`optimize inhibition of DPP-4. Various substituents on the
`xanthine core were scrutinized in a broad manner while a
`stronger emphasis was put on the residues at N-1, N-7, and
`C-8 because of their higher impact on activity. Common
`synthetic starting materials for the study were 8-chlorotheo-
`† Compound 1 has been deposited into the Protein Data Bank: PDB code
`phylline 3 and 8-bromoxanthine 7 (Schemes 1 and 2). Derivative
`2RGU.
`* To whom correspondence should be addressed. Phone: +49 7351
`syntheses starting from theophylline 3 were routinely carried
`54-5539/5013 or 7657. Fax: +49 7351 54-5181. E-mail: for M.E.,
`out first with alkylation of N-7 followed by nucleophilic
`matthias.eckhardt@boehringer-ingelheim.com; for E.L., elke.langkopf@
`displacement of the chlorine at C-8 by a diamine. The
`boehringer-ingelheim.com;
`for F.H.,
`frank.himmelsbach@boehringer-
`alkylations were conducted using an appropriate alkyl halide
`ingelheim.com.
`in the presence of a base. Displacement of the chlorine was
`# Department of Chemical Research.
`§ Department of Metabolic Diseases Research.
`performed employing an excess of the diamine to be introduced
`| Department of Lead Discovery.
`or the diamine in combination with potassium carbonate. In the
`¶ Department of Chemical Development.
`case of an asymmetric diamine, the nitrogen not to be reacted
`‡ Department of Drug Discovery Support.
`was preferentially embedded in tert-butyl carbamate, with
`⊥ Department of Drug Metabolism and Pharmacokinetics.
`subsequent release by treatment with acid. Employing 7 as the
`aAbbreviations: DPP-4, dipeptidyl peptidase 4; GLP-1, glucagon-like
`starting material followed a similar reaction sequence to
`peptide 1; GIP, glucose-dependent insulinotropic peptide.
`10.1021/jm701280z CCC: $37.00  2007 American Chemical Society
`Published on Web 12/01/2007
`
`Mylan EX 1005, Page 1
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`

`
`Letters
`
`Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6451
`
`Scheme 2. Syntheses of Compounds 6 and 1 a
`
`Table 1. DPP-4 Inhibitory Activity of Xanthines
`
`compd
`2
`5a
`5b
`5c
`6
`6ac
`6bc
`6ad
`6ae
`6af
`
`R1
`
`R7
`H
`CH2Ph
`CH2Ph
`Me
`CH2CHCMe2 Me
`CH2CCMe
`Me
`CH2Ph
`Me
`CH2CHCMe2 Me
`CH2CCMe
`Me
`CH2CHCMe2
`CH2Ph
`CH2CHCMe2
`(CH2)2Ph
`CH2CHCMe2
`CH2COPh
`
`X/Y
`NH/H
`NH/H
`NH/H
`NH/H
`CH2/NH2
`CH2/NH2
`CH2/NH2
`CH2/NH2
`CH2/NH2
`CH2/NH2
`
`DPP-4 IC50 (nM)
`3900
`2800
`580
`200
`82
`35
`88
`284
`56
`5
`
`Table 2. Effects of 8-(3-Aminopiperidin-1-yl)xanthines on DPP-4,
`hERG, and M1
`
`M1 IC50
`hERG
`DPP-4 IC50
`R1
`R7
`(%)a
`(nM)
`(nM)
`compd
`f
`(R)-6af
`25
`31
`6
`CH2CHCMe2
`(S)-6af
`f
`CH2CHCMe2
`50
`23
`3
`(R)-6ag
`g
`CH2CHCMe2
`5
`51
`4
`(S)-6ag
`g
`CH2CHCMe2
`26
`51
`2
`88b
`(R)-6bf
`f
`CH2CCMe
`6161
`4
`88b
`(S)-6bf
`f
`CH2CCMe
`1129
`9
`(R)-6bh
`h
`CH2CCMe
`518
`51
`8
`(R)-6bi
`i
`CH2CCMe
`1174
`88
`1
`(R)-6bj
`j
`CH2CCMe
`430
`78
`3
`1 [(R)]
`k
`CH2CCMe
`295
`97
`1
`a hERG current remaining at a test concentration of 1 µM. b Value of
`the racemic compound.
`
`Table 3. Selected Basic PK Data of Compound 1 in Rat and
`Cynomolgus Monkey
`
`species
`
`MRTtot,oral
`CL
`Foral
`t1/2,
`Vss
`(%)
`oral (h)
`(h)
`(L/kg)
`((mL/min)/kg)
`rata
`50.7
`35.9
`14.3
`5.4
`37.3
`monkeyb
`50.1
`41.4
`17.4
`15.8
`15.8
`a 5 mg/kg oral and intravenous dose. b 5 mg/kg oral and 1.5 mg/kg
`intravenous dose.
`
`enantiopure compounds bearing a dimethylallyl group on N-7.
`Unfortunately, both compounds show an unacceptably high
`inhibition of the hERG channel and affinity for the muscarinic
`receptor M1. By variation of the residue on N-1 based on the
`dimethylallylated xanthine, both side effects could not con-
`comitantly be reduced sufficiently while retaining the desired
`high inhibitory activity. These shortcomings also extended to
`compounds with bicyclic aryl- or heteroarylmethyl residues
`attached to N-1 such as in 6ag that were otherwise highly potent
`DPP-4 inhibitors. Switching to the 7-butynyl derivatized scaffold
`proved to be a very effective measure to essentially abolish
`hERG channel inhibition and reduce interaction with receptor
`M1 to a high degree. Various phenacyl and bicyclic aryl- and
`heteroarylmethyl residues attached to N-1 of this scaffold
`exhibited high DPP-4 inhibition with no perturbing interactions
`with the hERG channel or M1 receptor. Curiously, in the
`7-butynyl series the (R)-configured compounds were signifi-
`cantly more active in all cases assayed, contrasting with the
`results obtained from the 7-dimethylallyl series.
`
`a Reagents: (a) Me2CCHCH2Br or MeCCCH2Br, iPr2NEt, DMF, room
`temp, 8a 86%, 8b 86%; (b) R1-Hal, K2CO3, DMF, room temp, 77–98%;
`(c) (I) racemic, (R)- or (S)-3-Boc-aminopiperidine, K2CO3, DMF, 75 °C,
`67–96%; (II) TFA, DCM or HCl, iPrOH, 69–98%.
`b Compound bears chlorine instead of bromine at C-8.
`
`assemble the fully substituted xanthines. The preferred order
`of attachment of the three substituents was N-7 followed by
`N-1 and C-8, though the order of introduction of the last two
`substituents could be reversed to streamline the examination of
`the residue on N-1. Accordingly, treatment of xanthine 7 with
`different alkyl halides was conducted in the presence of a mild
`base, such as triethylamine or ethyldiisopropylamine, furnishing
`the N-7 derivatized xanthine 8 selectively; competing N-1
`alkylation was usually not observed. The next step, alkylation
`at N-1, was regularly performed using the stronger base
`potassium carbonate leading to 9. The ensuing nucleophilic
`substitution of the bromine at C-8 in xanthine 9 for the diamine,
`3-aminopiperidine, was preferably carried out using the N-tert-
`butyloxycarbonyl protected 3-aminopiperidine in the presence
`of potassium carbonate. The synthesis was concluded by the
`release of the amino functionality from its protection by
`treatment with acid. The enantiomerically pure compounds were
`obtained by reaction with the commercial enantiopure N-tert-
`butyloxycarbonyl protected 3-aminopiperidines.
`The DPP-4 inhibitory activity of the compounds was tested
`using a preparation of human DPP-4 derived from Caco-2 cells.
`The results obtained for the racemic xanthine derivatives in
`Schemes 1 and 2 are compiled in Table 1. Variations at N-7 or
`C-8 of 5a yielded 5b, 5c, and 6, all showing significantly
`increased DPP-4 inhibition. In particular, replacement of the
`piperazine at C-8 for the 3-aminopiperidine in 5a resulted in a
`tremendous increase in potency. Combining the structural
`features of these more potent compounds led to compounds 6ac
`and 6bc, which exhibited approximately a 100-fold and 50-
`fold higher inhibitory activity, respectively, than the original 2.
`Further optimization at N-1 based on the structure of 6ac
`resulted in an additional increase of potency when attaching
`the phenacyl group (6af), while the benzylated and the phen-
`ethylated 6ad and 6ae showed inferior activity.
`Further profiling was conducted using the pure enantiomers
`(R)-6af and (S)-6af (Table 2). DPP-4 inhibition of the (S)-
`configured compound was about twice as potent as of the (R)-
`configured one, a trend that was generally observed for
`
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`
`6452 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
`
`Letters
`
`Figure 1. Compound 1 (light-blue carbon atoms) bound to DPP-4.
`Active site residues Ser630, His740, and Asp708 are shown in orange.
`Three hydrogen bonds (shown by black dashes) are formed by the
`amino function on the piperidine ring with acceptor groups on the
`protein Glu205, Glu206, and Tyr662. A fourth hydrogen bond is formed
`between the C-6 carbonyl of the xanthine scaffold and the backbone
`amide of residue Tyr631. Aromatic stacking interactions are formed
`between the xanthine ring system and Tyr547 as well as between the
`quinazoline ring and Trp629.
`
`Figure 2. Inhibition of plasma DPP-4 activity after oral administration
`of 1 at 1 mg/kg in Wistar rats, Beagle dogs, and Rhesus monkeys.
`Data are represented as the mean ( SEM (n ) 5 for rats, n ) 3 for
`dogs and monkeys).
`
`Compound 1, showing potent DPP-4 inhibition in vitro and
`a low affinity for hERG channel and M1 receptor, has been
`further examined. A favorable crystalline modification of the
`free base of 1 has been produced that is characterized by a high
`melting point (202 °C) and high aqueous solubility at physi-
`ological pH value (pH 7.4, >5 g/L). Compound 1 displays a
`log D of 0.4 at pH 7.4 and pK a of 1.9 and 8.6 corresponding
`to the protonation of the quinazoline and the primary amino
`group, respectively.
`The X-ray crystal structure of 1 in complex with human
`DPP-4 allows one to depict the main interactions of the inhibitor
`within the enzyme active site and to rationalize the observed
`SAR (Figure 1). The aminopiperidine substituent at C-8 of the
`xanthine scaffold occupies the S2 subsite. Its primary amine
`forms a network of charge-reinforced hydrogen bonds to
`Glu205, Glu206, and Tyr662, amino acid residues that constitute
`the recognition site for the amino terminus of peptide substrates
`of DPP-4. The butynyl substituent at N-7 occupies the hydro-
`phobic S1 pocket of the enzyme. The xanthine moiety is
`positioned such that its uracil moiety lies on top of Tyr547,
`forming aromatic π-stacking interactions with the phenol of
`Tyr547. Thereby, the side chain of Tyr547 is pushed from its
`relaxed position in the uncomplexed enzyme.11 A similar
`
`Figure 3. Effect of 1 on plasma glucose levels in an oral glucose
`tolerance test in db/db mice (top). Compound or vehicle was admin-
`istered 45 min before an oral glucose load. Reactive plasma glucose
`AUC was calculated from 0 to 120 min (middle). Inhibition of plasma
`DPP-4 activity was measured 30 min after the glucose load at the peak
`of the glucose excursion. Data are represented as the mean ( SEM (n
`) 7/group).
`
`conformational change has been reported for related xanthine
`based inhibitors and for
`inhibitors from other structural
`classes.12–14 The C-6 carbonyl function of the xanthine scaffold
`forms a hydrogen bond to the backbone NH of Tyr631. Finally,
`the quinazoline subsituent at N-1 is placed on a hydrophobic
`surface patch of the protein and interacts with Trp629 by
`π-stacking its phenyl ring with the pyrrol ring of the amino
`acid side chain.
`The observed boost in affinity upon the introduction of the
`aminopiperidine group at C-8 is due to the very intimate
`interaction of the positively charged terminal ammonium that
`can form three strong hydrogen bonds with the protein. Further,
`the observed bound chair conformation of the piperidine ring
`is a low-energy conformation. The original piperazine derivative
`2 can only form two hydrogen bonds and needs to adopt an
`unfavorable conformation upon binding.12
`The strong DPP-4 inhibition of 1 has been confirmed in
`various species in vitro and in vivo. In male Wistar rats, Beagle
`dogs, and Rhesus monkeys, xanthine 1 proved to be a highly
`efficacious, long-lasting, and potent DPP-4 inhibitor providing
`>70% inhibition for >7 h for all three species after oral
`administration of 1 mg/kg (Figure 2).
`Pharmacokinetic parameters including oral bioavailability,
`clearance, mean residence time, and volume of distribution of
`1 in rat and Cynomolgus monkey are summarized in Table 3.
`The long terminal half-life and high volume of distribution are
`
`Mylan EX 1005, Page 3
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`

`
`Letters
`
`Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6453
`
`key characteristics of 1 and, in combination with its high potency
`and good oral bioavailability, are thought to contribute to the
`strong and long-lasting inhibitory effect on DPP-4 observed in
`vivo.
`Compound 1 was further characterized in vivo in diabetic
`mice (Figure 3). Single oral administration of 1 to db/db mice
`45 min prior to an oral glucose tolerance test reduced plasma
`glucose excursion in a dose-dependent manner from 0.1 mg/kg
`(15% inhibition) to 1 mg/kg (66% inhibition). The improvement
`of oral glucose tolerance correlated with the DPP-4 activity in
`plasma, which was inhibited by 76% with the 1 mg/kg dose 30
`min after the glucose load was administered.
`Compound 1 exhibits no interaction with CYP-450 enzymes
`up to 50 µM. Because inhibition of DPP-8 and DPP-9, which
`are closely related to DPP-4, has been associated with toxicities
`in animals, it is important to note that 1 displays a more than
`10000-fold selectivity against both of these enzymes.15
`In summary, a new chemical class of highly potent DPP-4
`inhibitors structurally based on the xanthine scaffold has been
`discovered. The 3-aminopiperidine attached to C-8 proved to
`be a crucial constituent for high inhibitory activity, and but-2-
`ynyl on N-7 was essential to eliminate interaction with the hERG
`channel and M1 receptor. Further optimization led to 1 bearing
`a quinazolin-2-ylmethyl at N-1. 1 represents a highly potent,
`selective, and long-acting DPP-4 inhibitor of a novel chemotype
`that shows promise for once-daily treatment of type 2 diabetic
`patients. Compound 1 is currently undergoing clinical phase
`IIb trials.
`
`Acknowledgment. The authors are indebted to their associ-
`ates for their dedicated and excellent technical assistance.
`
`Supporting Information Available: Experimental details, ana-
`lytical data of the compounds, and X-ray crystallograpic data of 1.
`This material is available free of charge via the Internet at http://
`pubs.acs.org.
`
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