`M E M O
`P h a r m a c e u t i c a l R & D - R a h w a y
`TO: Tim Rhodes
`
`FROM: Leigh Shultz
`
`DATE: 30 Jun 2004
`
`CONTRIBUTORS: Alex Chen, Russell Ferlita, Robert Wenslow, Dina Zhang, James Qin, Peter Dormer,
`Leonardo Allain, Jenna Leschek, Chris Lindemann
`
`SUBJECT: Physico-chemical characteristics of L-000224715 phosphate salt, monohydrate form
`
`L-000224715, also known as MK-0431, is a DPP-IV inhibitor for the treatment of Type II Diabetes Mellitus. It
`was approved for development as a PCC by SARC in January of 2002 and began Phase I clinical trials in July
`2002. Phase I and initial Phase II clinical supplies were manufactured with Forms I and III of the anhydrous
`phosphate salt; a monohydrate was discovered in March 2003, and after analysis of its properties, the decision
`was made to use the monohydrate for the Phase III and market formulations. This memo describes the chemical
`and physical properties of the monohydrate form of the monobasic phosphate salt. Polymorph and physical form
`data contained in this memo supercedes that found in the L-224715 Preformulation Report issued in Sep 2002.
`
`cc: MK-0431 IDT, S. Karki
`
`Merck Exhibit 2149, Page 1
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`IPR2020-00040
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`

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`Table of Contents
`1.0 SUMMARY............................................................................................................................................................. 3
`
`2.0 DESCRIPTION.......................................................................................................................................................3
`2.1 NAME, STRUCTURE, FORMULA............................................................................................................................................................3
`2.2 COLOR, FORM, APPEARANCE...............................................................................................................................................................3
`3.0 TEST SUBSTANCES............................................................................................................................................. 3
`
`4.0 PHYSICAL CHARACTERIZATION..................................................................................................................4
`4.1 MICROSCOPY.............................................................................................................................................................................................. 4
`4.2 DIFFERENTIAL SCANNING CALORIMETRY (DSC)................................................................................................ 5
`4.3 THERMOGRAVIMETRIC ANALYSIS (TGA)............................................................................................................ 6
`4.4 X-RAY POWDER DIFFRACTION (XRPD)...............................................................................................................7
`4.5 SOLID STATE NMR (SSNMR)............................................................................................................................. 8
`4.6 CRYSTAL FORMS/POLYMORPHISM.....................................................................................................................................................9
`4.7 HYGROSCOPICITY...................................................................................................................................................................................11
`4.8 EQUILIBRIUM SOLUBILITY IN AQUEOUS MEDIA........................................................................................................................ 12
`4.9 EQUILIBRIUM SOLUBILITY IN ORGANIC MEDIA......................................................................................................................... 12
`4.10 PKA............................................................................................................................................................................................................13
`4.11 DISSOLUTION AND SOLUTION PROPERTIES................................................................................................................................13
`4.12 UV/VIS ABSORBANCE SPECTRUM 13
`5.0 STABILITY...........................................................................................................................................................15
`5.1 BULK DRUG STABILITY....................................................................................................................................................................... 15
`5.2 SOLUTION STABILITY............................................................................................................................................................................16
`5.3 MODES OF DEGRADATION.................................................................................................................................................................. 17
`6.0 ANALYTICAL METHODS................................................................................................................................ 18
`6.1 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY....................................................................................................................18
`6.2 HPLC/MS/MS....................................................................................................................................................18
`6.3 UV/VIS ABSORBANCE..........................................................................................................................................................................18
`6.4 SOLID-STATE NMR............................................................................................................................................ 19
`
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`1.0 SUMMARY
`
`This memo summarizes the physico-chemical characterization of the crystalline monobasic monohydrate form of the
`phosphate salt of L-000224715 (MK-0431). The monohydrate form is a stable, high-melting, non-hygroscopic material with
`four known anhydrous polymorphs and multiple isomorphic solvates. It is well-soluble in aqueous media across the
`physiological pH range and has adequate stability in solution below pH 6.
`
`2.0 DESCRIPTION
`
`L-000224715, a DPP-IV inhibitor, is being developed for oral administration as a treatment for Type II (adult-onset)
`Diabetes Mellitus (T2DM).
`
`2.1 Name, Structure, Formula
`
`L-000224715 has a molecular weight of 407.321 g/mol and a molecular formula of C16H15F6N5O. The monohydrate form of
`the monobasic phosphate salt, has a molecular weight of 523.332 g/mol (salt factor 1.285) and the molecular formula
`C16H20F6N5O6P. The structure of L-000224715 is shown in Figure 1, below.
`F
`
`F
`
`F
`
`NH2
`
`O
`
`N
`
`N
`
`N
`
`N
`
`CF3
`
`Figure 1. Structure of L-000224715
`
`2.2 Color, Form, Appearance
`
`The monohydrate phosphate salt of L-000224715 is a white, crystalline powder with rod-like individual crystals.
`
`3.0 TEST SUBSTANCES
`
`All experiments were performed on lots of monohydrate phosphate salt (L-000224715-010X) provided by Chemical
`Engineering R&D. XRPD data were obtained on all lots to confirm form; specific lots for data shown in figures are noted in
`the text. Thermal characterization (DSC, TGA) was performed on lot 66839-113. Both hygroscopicity and stability data
`were obtained with lot 66839-123; solubility numbers were obtained with material from lot 66839-142. Some of the data in
`the polymorph/solvate section was obtained with a lot of the anhydrous material, 006F007. Data obtained from other lots of
`phosphate salt are noted in the text. Both aqueous solubility as a function of pH and the pKa were determined with the
`crystalline free base, L-000224715-000T001.
`
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`4.0 PHYSICAL CHARACTERIZATION
`
`4.1 Microscopy
`
`Optical microscopy performed on lot L-000224715-66839-142 at 100X magnification (Figure 2) reveals a birefringent,
`crystalline material. The primary crystals have a well-defined rod-like morphology.
`
`Figure 2. Optical microscopic image of L-000224715 phosphate salt
`monohydrate (lot 66839-142). Average crystal length is 250 m.
`
`Two SEM images of a representative lot of L-000224715-010X are shown in Figure 3. The lamellar nature of the individual
`crystals is evident in this image.
`
`Figure 3. SEM images of L-000224715 phosphate salt
`monohydrate crystals [76x (left) and 2293x (right)
`magnification]
`
`Figure 4 shows particle size data for four lots of L-000224715 monohydrate and one lot of anhydrous material, obtained
`using a MicroTrac light-scattering instrument with isopropanol as the medium. Lot 113 shows a broad, unimodal particle
`size distribution with a mean of 143 m after 30 s of sonication (D10 = 52 m, D95 = 329 m). Prior to sonication, the mean
`particle size was 198 m, with a D10 of 77 m and a D95 of 422 m. The decrease in both D95 and the mean on sonication
`indicates the friability of the crystals shown in Figure 3. Lots 125 and 144 have similar average particle sizes of 165 and 136
`m, respectively. A representative lot of the anhydrous phosphate salt (lot 006F024, plate-like morphology) had an average
`particle size of 77 m after 30 s of sonication.
`
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`L-224715
`Sonication = 30 s
`Monohydrate
` NB# 66839-113
` NB# 66839-125
` NB# 66839-138
` NB# 66839-144
`Anhydrate
` lot24
`
`10
`
`8 6 4 2 0
`
`% Frequency
`
`1
`
`10
`
`100
`Particle Size ( m)
`
`1000
`
`Figure 4. Comparison of particle size distribution for four lots of L-000224715 phosphate salt
`monohydrate and one lot of anhydrous material
`
`4.2 Differential Scanning Calorimetry (DSC)
`
`A DSC thermogram for lot 66839-142 is shown in Figure 5. Data were obtained by heating a sample in a closed pan at 10
`°C/min. The endothermic transition with a maximum at 140.4 °C is due to loss of crystalline water. An endotherm due to
`melting of Form I is observed at 209.16 °C (onset) with an enthalpy of 243.2 J/g. The endotherm cannot be reliably
`quantitated due to exothermic decomposition immediately following melting. When the monohydrate is held at 90 °C for 1
`hour in the DSC cell (Figure 6), the dehydrated monohydrate is formed. Subsequent heating of the sample at 5 °C/min
`results in transitions related to formation of a metastable anhydrous form (IV) and then Form I.
`
`Figure 5. DSC thermogram of L-000224715, lot 66839-142
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`Figure 6. Enlargement of the initial region of the DSC thermogram for the dehydrated
`monohydrate, showing the endothermic and exothermic transitions to Forms IV and I, respectively
`
`4.3 Thermogravimetric Analysis (TGA)
`
`A TGA trace for L-000224715 phosphate salt monohydrate is shown in Figure 7. A sample heated at 10 °C/min under
`nitrogen flow shows a weight loss of 3.4% up to 200 °C. This weight loss corresponds to loss of one molar equivalent of
`crystalline water from the lattice. Figure 8 shows data from an isothermal TG experiment indicating the rate of water loss
`from the monohydrate at 60 °C. Under nitrogen flow (very low relative humidity) at elevated temperature, all of the
`crystalline water is lost within 6 hours to form the dehydrated monohydrate and Form IV, which convert rapidly back to the
`monohydrate under ambient conditions.
`
`Figure 7. TGA trace of L-000224715 phosphate salt monohydrate
`
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`Figure 8. Isothermal TG results indicating the rapid rate of water loss at 60 °C under nitrogen flow (within 400 min). Water
`is rapidly absorbed by the sample when it is cooled to 25 °C.
`
`4.4 X-ray Powder Diffraction (XRPD)
`
`The X-ray powder diffraction pattern for the monohydrate form of the phosphate salt of L-000224715 between 4 and 40° 2
`is shown in Figure 9. Numerous sharp reflections are observed in this region, indicating the high degree of crystallinity of
`the monohydrate.
`
`counts
`
`25600
`
`19600
`
`14400
`
`10000
`
`6400
`
`3600
`
`1600
`
`400
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`°2Theta
`
`Figure 9. XRPD pattern for L-000224715-66839-123, phosphate salt monohydrate
`
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`4.5 Solid-State NMR (SSNMR)
`
`Solid-state NMR has been useful, especially in conjunction with XRPD and thermal techniques, to characterize the solid
`phases of L-000224715 phosphate salt. 13C CP-MAS NMR can be used to distinguish between forms and to determine the
`presence of organic solvates, while use of the 19F nuclei for characterization allows SSNMR to be used for characterization of
`drug-excipient blends and dosage forms, since none of the common excipients contain fluorine. All 19F, 13C, and 31P spectra
`for L-000224715 were obtained using standard pulse sequences. 31P solid-state NMR is also useful and less time-
`consuming for characterization and differentiation of the forms of the phosphate salt. 13C, 19F, and 31P spectra of the
`monohydrate form of the phosphate salt are shown in Figures 10, 11, and 12, respectively.
`
`300
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`
`Figure 10. 13C CP-MAS NMR spectrum of L-000224715 phosphate salt monohydrate
`
`-50
`
`-100
`
`-150
`
`-200
`
`Figure 11. 19F MAS NMR spectrum of L-000224715 phosphate salt monohydrate
`
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`20
`
`10
`
`0
`
`-10
`
`-20
`
`Figure 12. 31P MAS NMR spectrum of L-000224715 phosphate salt monohydrate
`(high-power 1H decoupling)
`
`4.6 Crystal Forms/Polymorphism
`
`The monohydrate form of the phosphate salt of L-000224715 can be crystallized directly from aqueous solution but is most
`easily isolated from IPA/water or ISAA (isoamyl alcohol)/water systems. Heating the monohydrate to temperatures between
`40 and 130 °C under nitrogen results in formation of a dehydrated monohydrate phase having a similar XRPD pattern but
`distinguishable by its lack of TG weight loss. This transformation is fully reversible, as the monohydrate is re-formed upon
`exposure to ambient laboratory conditions. Continued heating of the dehydrated monohydrate phase will result in formation
`of anhydrous Form IV, which can be turned over to the monohydrate in aqueous slurries or in the solid state at relative
`humidities above 98% (25 °C). Form IV is a monotropic polymorph of anhydrous Form I, which is the most
`thermodynamically stable of the anhydrous forms. Form I readily forms the monohydrate in aqueous slurries. The solid
`phases present in the monohydrate system are summarized in Figure 13 below.
`
`RT
`
`40130 °C
` N2 flow
`
`Monohydrate
`
`Dehydrated
`monohydrate
`
`60130 °C
`
`Form IV
`(anhydrous)
`
`130 ~ 180 °C
`
`Form I
`
`RH=98%, RT
`
`Water slurry with
`or without
`monohydrate seed
`
`Figure 13. Solid phases in the monohydrate system
`
`Prior to the discovery of the monohydrate, deliveries of the phosphate salt were mixtures of anhydrous Form I and anhydrous
`Form III. Form I/III mixtures can be isolated indirectly from IPA/water or EtOH water systems by crystallization of the IPA
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`or EtOH solvate followed by drying to produce Form II, a desolvated solvate. Further drying produces mixtures of Forms I
`and III, and conversion to mostly Form III is observed on milling and/or compaction of the anhydrous material. Forms I and
`III are enantiotropic polymorphs; Form III is the most stable form below the transition temperature of 34 °C. A polymorph
`map of the anhydrous phases of L-000224715 is shown in Figure 14.
`
`Form II
`
` RT
`
`Form I
`
`Agitation
`
`Form III
`
`Ttrans = 34 C
`Figure 14. Solid phases in the anhydrous system
`
`Figure 15 shows 19F SSNMR spectra for the phases observed in the phosphate salt system, including monohydrate and
`anhydrous phases.
`
`Form I
`
`Form II
`
`Mostly Form III
`
`Form IV
`From Monohdyrate
`
`EtOH Solvate
`
`Monohydrate
`
`-90
`-100
`-110
`-120
`Figure 15. 19F SSNMR spectra of the solid phases of L-000224715 phosphate salt
`
`-130
`
`The solid phases in the monohydrate system (see map in Figure 13) can be produced by heating a sample of the monohydrate
`on the hot-stage of an X-ray powder diffraction instrument. The resulting XRPD patterns for the dehydrated monohydrate,
`Form IV, and Form I are shown in Figure 16 below. Prior to discovery of the monohydrate, a number of solvates were
`known to be produced from slurries of the anhydrous forms in various organic solvents. The XRPD patterns for some of
`these solvates are shown in Figure 17 for purposes of illustration (methanol, ethanol, isopropanol, acetonitrile, ethyl acetate
`(EtOAc), acetone, N,N-dimethylformamide (DMF), and 1-octanol).
`
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`Figure 16. XRPD patterns for solid phases of L-000224715 in the monohydrate system
`
`Figure 17. XRPD patterns for solvates of L-000224715, prior to the discovery of the monohydrate form (note slurrying in
`water did not produce monohydrate at that time)
`
`Amorphous material can be made by lyophilization of an aqueous solution, and this amorphous material is physically stable
`at 20 °C. Small amounts of the lyophilized material will recrystallize to Form I when exposed to heat or humidity; samples
`recrystallized within days at 40 °C/75% RH, within minutes on heating in the DSC, and within hours upon exposure to
`~65% RH on the moisture sorption balance at 40 °C. It should be noted that these experiments with the amorphous material
`were carried out prior to the discovery of the monohydrate form.
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`4.7 Hygroscopicity
`
`The hygroscopicity of monohydrate lot L-000224715-66839-123 was determined at 25 and 40 °C (Figure 18a and b) from 5
`to 95% RH using a dynamic vapor sorption balance (VTI SGA-100). At both temperatures, the solids were not dried prior to
`the hygroscopicity experiment. At 25 °C, they gain less than 1% water by weight at 75% RH; at 40 °C, they gain less than
`0.3% water by weight. The material is non-hygroscopic at both 25 and 40 °C, and no changes in form were observed after
`the experiments. No water loss from the monohydrate was observed in either experiment, even at relative humidities as low
`as 0.8%; long equilibration times were used to ensure equilibrium was reached at each RH step.
`Adsorption/Desorption Isotherm
`L-224715 monohydrate (KF=3.3%)
`Lot 66839-123
`
`Adsorption
`Desorption
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`70
`
`80
`
`90
`
`100
`
`%RH
`
`Adsorption/Desorption Isotherm
`L-224715 monohydrate, 40 C
`
`5.00
`
`4.00
`
`3.00
`
`2.00
`
`1.00
`
`0.00
`
`-1.00
`
`Weight (% change)
`
`2.593
`
`Adsorption
`Desorption
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`0.260
`70
`80
`
`90
`
`100
`
`%RH
`
`3.0
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`-0.5
`
`Weight (% change)
`
`Figure 18. Hygroscopicity of L-000224715-66839-123 at 25 °C (a) and 40 °C (b)
`
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`4.8 Equilibrium Solubility in Aqueous Media
`
`The solubility of the monohydrate was determined using the shake-flask method at 22 °C (drug lot 66839-142). The
`solubility of the monohydrate phosphate salt in water was found to be 63.8 mg/mL L-000224715 with a native pH of 4.5.
`Solubility data were also obtained at pH 3.2, 4.1, and 7.1 in buffers (Table 1). To obtain additional data, solubility for
`L-000224715 was obtained across the physiological pH range by titration of 50 mg of the crystalline free base,
`L-000224715-000T001, with 10-mol% aliquots of HCl (Figure 19). At pH 8, the solubility was 12 mg/mL; this increased to
`40 mg/mL at pH 7.4 and to more than 45 mg/mL at pH 7, where the entire sample had dissolved.
`
`Table 1. Solubility of L-000224715 in various aqueous systems
`
`Solution (form)
`Water, 22 °C (monohydrate)
`Water, 5 °C (monohydrate)
`0.01 M HCl (monohydrate)
`0.10 M sodium citrate (monohydrate)
`0.10 M sodium carbonate (free base)
`
`Final pH Solubility of L-000224715 (mg/mL)
`4.5
`63.8
`4.4
`58.2
`3.2
`68.1
`4.1
`66.1
`7.1
`42.2
`
`Solubility of L-000224715 as a function of pH
`
`7.00
`
`8.00
`pH
`
`9.00
`
`10.00
`
`50.00
`45.00
`40.00
`35.00
`30.00
`25.00
`20.00
`15.00
`10.00
`5.00
`0.00
`6.00
`
`[L-000224715] (mg/mL)
`
`Figure 19. Aqueous solubility of L-000224715 as a function of pH (titration data)
`
`L-000224715 is very soluble across the physiological pH range. Suspension of anhydrous Form I in water at 22 °C resulted
`in conversion to the monohydrate and a measured solubility of 62.2 mg/mL. Although the solubility of the monohydrate is
`lower than that of the anhydrous forms, solubility is not expected to limit bioavailability for this compound.
`
`4.9 Equilibrium Solubility in Organic Media
`
`The equilibrium solubility of L-000224715-66839-142 was also determined in possible granulating and coating solvents
`(acetone, isopropanol, and 90/10 v/v IPA/water). The solubility data are shown in Table 2 along with the resulting form in
`the slurry.
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`Table 2. Solubility of L-000224715-66839-142 in organic media
`
`Solvent
`
`acetone
`2-propanol
`90% (v/v) 2-PrOH
`
`Solubility
`(mg L-224715/mL)
`0.5
`0.14
`0.69
`
`Form by XRPD
`
`solvate
`solvate
`monohydrate
`
`See Section 4.6 for discussion of the solvates. Analysis of the supernatants from the acetone and IPA samples indicated that
`some degradation had taken place.
`
`4.10 pKa
`
`The pKa of L-000224715 was determined in water by potentiometric titration of the free base. The average value
`determined, 8.03
` 0.06, is attributed to the primary amine moiety and is in good agreement with the value of 7.5 determined
`from the solubility titration of the free base (see Section 4.9).
`
`4.11 Dissolution and Solution Properties
`
`Although the monohydrate phosphate salt of L-000224715 is non-hygroscopic, it is highly soluble in water and dissolves
`rapidly upon contact with water. It also goes into solution rapidly in acidic media such as 0.1 N HCl and 0.1% H3PO4.
`Water and acidic solution are both suitable as a dissolution media for L-000224715. Intrinsic dissolution data from the
`different anhydrous forms and the monohydrate are shown in Figure 20. The monohydrate is slightly slower to dissolve but
`does not affect in vitro or in vivo performance of an IR dosage form.
`
` Form I
` Form II
` Form III
` Monohydrate
`
`80x10-3
`
`60
`
`40
`
`20
`
`0
`
`Concentration (mg/ml)
`
`5
`
`10
`
`15
`20
`Time (minute)
`Figure 20. Intrinsic dissolution data for the major forms of L-000224715
`
`25
`
`30
`
`4.12 UV/Vis Absorbance Spectrum
`
`The UV/Visible spectrum of L-000224715 was determined in 50% (v/v) acetonitrile (Figure 20). Absorption maxima are
`observed at 202 and 267 nm.
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`UV-Visible Spectrum of L-000224715
`
`267
`
`200
`
`0.195
`
`0.095
`
`-0.005
`
`Absorbance (AU)
`
`190 200 210 220 230 240 250 260 270 280 290 300 310 320
`Wavelength / nm
`
`Figure 21. UV-Visible absorbance spectrum of L-000224715 (50% acetonitrile in water)
`
`5.0 STABILITY
`
`5.1 Bulk Drug Stability
`
`The bulk stability of a sample of wet-milled phosphate salt monohydrate was assessed at 1, 2, and 4 weeks at 40 and 80
`°C/amb RH and 40 °C/75% RH. Stability data are reported as % initial claim (Table 3) and as area % (Table 4) relative to
`samples kept at 20 °C. No degradation was detected in any of the samples. Physical stability studies conducted in parallel
`suggested that some reversible dehydration to form the dehydrated monohydrate was occurring at 80 °C on stability (as
`determined by TG and XRPD).
`
`Table 3. Solid-state stability (% initial claim) of L-000224715-66839-123 (wet-milled)
`
`Station
`
`40 °C/amb RH
`40 °C/75% RH
`80 °C/amb RH
`
`% Initial Claim L-000224715
`2 wks
`102.0
`102.0
`1018
`
`4 wks
`103.6
`106.8
`99.7
`
`1 wk
`96.8
`98.0
`97.8
`
`Table 4. Solid-state stability (relative area %) of L-000224715-66839-123 (wet-milled)
`
`
`
`Relative Area % L-000224715
`2 wks
`4 wks
`100.0
`100.0
`100.0
`100.0
`100.0
`100.0
`
`1 wk
`100.0
`100.0
`100.0
`
`
`
`No photodegradation of any of the forms of L-000224715 phosphate salt has been noted.
`
`Station
`
`40 °C/amb RH
`40 °C/75% RH
`80 °C/amb RH
`
`U:\L224715\L-224715 phosphate salt monohydrate MEMO.doc
`
`15
`
`Merck Exhibit 2149, Page 15
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`5.2 Solution Stability
`
`The solution stability of L-000224715 was assessed in solution at 40 and 80 °C in 20 mM buffers (pH 4, acetate; pH 6 and 8,
`phosphate; pH 10, carbonate), 0.01 N HCl, and deionized water at 0.1 mg/mL. Data are reported as area percents relative to
`samples stored at 20 °C (Table 5).
`
`Table 5. Solution thermal stability of L-000224715
`
`Conditions
`
`water
`pH 2
`pH 4
`pH 6
`pH 8
`pH 10
`
`Relative Area % L-224715, 40 °C
`1 wk 2 wk 4 wk
`93.9
`89.8
`75.9
`99.9
`98.1
`100.6
`101.8
`93.2
`94.1
`99.0
`101.6
`98.5
`88.2
`81.6
`60.2
`83.1
`62.9
`36.5
`
`Relative Area % L-224715, 80 °C
`1 wk 2 wk 4 wk
`0.0
`0.0
`0.0
`99.0
`100.3
`95.4
`100.9
`93.6
`90.9
`24.9
`5.0
`0.4
`0.0
`0.0
`0.0
`0.0
`0.0
`0.0
`
`From this initial study, it was observed that L-000224715 is most stable at pH 2 and 4 and that it degrades rapidly at elevated
`temperatures (80 versus 40 °C) and in basic solution. A more extensive solution study was done with L-000224715 in
`solution from pH 1 to pH 10 at 40 °C. Pseudo-first order rate constants were calculated (with units of 1/h) for loss of
`L-000224715 with time and are graphed logarithmically in Figure 22.
`Rate of Degradation vs pH, 40 C
`
`-2.250
`-2.750
`-3.250
`-3.750
`-4.250
`-4.750
`-5.250
`-5.750
`
`log k(obs)
`
`0
`
`2
`
`4
`
`6
`
`pH
`
`8
`
`10
`
`Figure 22. pH-rate profile for degradation of L-000224715 in solution at 40 °C
`
`As a comparison, the half-life for degradation at pH 4 is 13.5 years, at pH 7 is 10 weeks, and at pH 10 is 7.4 days. Acid-
`catalyzed degradation is observed at pHs lower than 4; both thermal and base-catalyzed degradation are observed at high pH.
`The data below pH 4 are cleaner due to a single pathway for degradation. Two modes of degradation are observed at higher
`pH and cause more scatter in the data.
`
`U:\L224715\L-224715 phosphate salt monohydrate MEMO.doc
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`16
`
`Merck Exhibit 2149, Page 16
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`5.3 Modes of Degradation
`
`The degradation of L-000224715 observed in solution was studied by LC/MS/MS. The proposed degradation pathways
`elucidated in this study are shown in Scheme 1, below.
`
`Scheme 1. Solution degradation pathways for L-000224715
`
`F
`
`F
`
`F
`
`F
`
`F
`
`F
`
`NH2
`
`O
`
`L-224715
`
`N
`
`N
`
`base
`or
`acid
`
`N
`
`N
`
`CF3
`
`O
`
`N
`
`N
`
`N
`
`N
`
`CF3
`
`F
`
`F
`
`F
`
`F
`
`F
`
`F
`
`NH2
`
`O
`
`HN
`
`O
`
`N
`
`N
`
`N
`
`CF3
`
`amide bond cleavage
`degradates
`
`O
`
`N
`
`N
`
`N
`
`N
`
`CF3
`
`elimination (de-amination) degradates
`
`The main path for degradation is hydrolysis of the amide bond, which proceeds by both an acid- and base-catalyzed
`mechanism to produce a carboxylic acid and a free amine, which is UV silent above 220 nm. L-000224715 also degrades
`thermally by an elimination mechanism to produce a mixture of unsaturated products. The thermodynamic product,
`identified by solution 1H NMR (P. Dormer, Process Research), is the trans olefin in conjugation with the aryl ring. The trans
`olefin in conjugation with the amide has been identified as a minor product. The formation of the elimination products is
`accelerated by base, which aids in deprotonation. At very high pH, however (pH 10), hydrolysis predominates.
`
`U:\L224715\L-224715 phosphate salt monohydrate MEMO.doc
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`17
`
`Merck Exhibit 2149, Page 17
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`6.0 ANALYTICAL METHODS
`
`6.1 High Performance Liquid Chromatography
`
`System:
`Column:
`Mobile phase:
`Flow:
`Detector:
`Temperature:
`Inj. vol:
`Diluent:
`Sample conc.
`Gradient:
`
`Perkin Elmer Series200
`YMC-Pack ODS-AM 5 m 250X4.6mm
`A) 0.1% phosphoric acid B) acetonitrile
`1.0 mL/min
`210 nm
`ambient
`20 L
`0.1% phosphoric acid
`0.1 mg/mL of L-000224715
`
`Time
`(min)
`0
`2
`13
`16
`25
`27
`28
`36
`
`A
`
`98
`98
`59
`59
`30
`30
`98
`98
`
`B
`
`2
`2
`41
`41
`70
`70
`2
`2
`
`L-000224715 has a retention time of 14.8
`
` 0.1 min; the des-fluoro process impurities elute at 14.2
`
` 0.1 and 14.4
`
` 0.1 min.
`
`6.2 HPLC/MS/MS
`
`Stability samples of L-000224715 containing degradates were analyzed by LC/MS/MS (J. Qin) to determine the degradate
`molecular weights. The LC/MS/MS method is as follows:
`
`Waters 2790
`LC System:
`Symmetry C18 5 m 150X3.9 mm
`Column:
`A) 0.1% formic acid B) acetonitrile
`Mobile phase:
`0.2 mL/min
`Flow:
`Micromass Q-TOF2 (ESI)
`Detector:
`Ionization mode: Positive
`Temperature:
`40 °C
`Inj. vol:
`50 L
`Sample conc.
`0.1 mg/mL of L-000224715
`Gradient:
`
`A
`
`B
`
`Time
`(min)
`0
`15
`
`95
`10
`
`5
`90
`
`6.3 UV/Vis Absorbance
`
`The absorbance spectrum was obtained in 50% acetonitrile/water (quartz cuvette) on an Agilent 8453 UV/Vis spectrometer
`by Leonardo Allain (PAC). The concentration of the API in solution was 0.0081 mg/mL.
`
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`18
`
`Merck Exhibit 2149, Page 18
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`6.4 Solid-state NMR
`
`The solid-state 13C NMR spectrum was obtained by Robert Wenslow (Analytical Research) on a Bruker DSX 400WB NMR
`system using a Bruker 4-mm double resonance CPMAS probe. The 13C NMR spectrum utilized 1H/13C cross-polarization
`magic-angle spinning with variable-amplitude cross polarization. The sample was spun at 15.0 kHz, and a total of 2048
`scans were collected with a recycle delay of 20 seconds. A line broadening of 40 Hz was applied to the spectrum before FT
`was performed. Chemical shifts are reported on the TMS scale using the carbonyl carbon of glycine (176.03 ppm) as a
`secondary reference.
`
`The solid-state 19F NMR spectrum was obtained on a Bruker DSX 400WB NMR system using a Bruker 4-mm CRAMPS
`probe. The NMR spectrum utilized a simple pulse-acquire pulse program. The samples were spun at 15.0 kHz, and a total of
`16 scans were collected with a recycle delay of 30 seconds. A vespel endcap was utilized to minimize fluorine background.
`A line broadening of 100 Hz was applied to the spectrum before FT was performed. Chemical shifts are reported using
`poly(tetrafluoroethylene) (Teflon®) as an external secondary reference which was assigned a chemical shift of 122 ppm.
`
`The solid-state 31P NMR spectrum was obtained on a Bruker DSX 400WB NMR system using a Bruker 4-mm CPMAS
`probe. The NMR spectrum utilized a simple pulse-acquire pulse program with high power 1H decoupling. The samples were
`spun at 15.0 kHz, and a total of 128 scans were collected with a recycle delay of 5 seconds. A line broadening of 100 Hz was
`applied to the spectrum before FT was performed. Chemical shifts are reported using phosphoric acid as an external
`secondary reference which was assigned a chemical shift of 0 ppm.
`
`U:\L224715\L-224715 phosphate salt monohydrate MEMO.doc
`
`19
`
`Merck Exhibit 2149, Page 19
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`