Solvates of Zwitterionic Rifampicin: Recurring ... - ACS Publications

Subscriber access provided by READING UNIV

Article

Solvates of zwitterionic rifampicin: recurring packing motifs via nonspecific interactions Barbara Wicher, Krystian Pyta, Piotr Przybylski, and Maria Gdaniec Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01121 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on January 6, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Crystal Growth & Design is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

Solvates of zwitterionic rifampicin: recurring packing motifs via nonspecific interactions Barbara Wicher,*,† Krystian Pyta,‡ Piotr Przybylski‡ and Maria Gdaniec*,‡

†Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznań, Poland, ‡Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznań, Poland Antibiotic rifampicin was recrystallized from solutions containing aliphatic alcohols, ketones or DMSO. Thirteen new solvates were obtained that were studied by X-ray crystallography. In these solvates rifampicin exists in a zwitterionic form. The reported solvates are classified into eight different structural types. Whereas rifampicin molecules in the solvates form diverse assemblies via hydrogen bonds, the recurring structural motif in these crystals is generated by nonspecific intermolecular interactions. This motif has the form of a periodic 1D rod and similar 1D motifs were found in the structures of other zwitterionic or ionic rifamycins. In rifampicin solvates, these rods form a basis for the construction of two types of 2D periodic modules: monolayers found in six structural types of rifampicin solvates and bilayers observed in two types of solvates. Diverse mutual arrangements of these 2D modules lead to the formation of channels with accommodated solvent molecules that consist 10-40% of the crystal volume. DTA/TG analyses and powder X-ray diffraction show that desolvation of the rifampicin 2-propanol solvate results in polymorph II of rifampicin. We show here that solvate decomposition may be a route to transformation of a thermodynamically stable polymorphic form into a metastable one.

e-mail: [email protected]; Tel: +48 61 8291684 ; e-mail: [email protected]; Tel: +48 61 8546436

ACS Paragon Plus Environment

Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 38

Solvates of zwitterionic rifampicin: recurring packing motifs via nonspecific interactions Barbara Wicher,*,† Krystian Pyta,‡ Piotr Przybylski,‡ and Maria Gdaniec*,‡ †Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznań, Poland, ‡ Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznań, Poland

KEYWORDS: rifampicin, solvates, crystal structure, intermolecular interactions, polymorphism

ABSTRACT: Antibiotic rifampicin was recrystallized from solutions containing aliphatic alcohols, ketones or DMSO. Thirteen new solvates were obtained that were studied by X-ray crystallography. In these solvates rifampicin exists in a zwitterionic form. The reported solvates are classified into eight different structural types. Whereas rifampicin molecules in the solvates form diverse assemblies via hydrogen bonds, the recurring structural motif in these crystals is generated by nonspecific intermolecular interactions. This motif has the form of a periodic 1D rod and similar 1D motifs were found in the structures of other zwitterionic or ionic rifamycins. In rifampicin solvates, these rods form a basis for the construction of two types of 2D periodic modules: monolayers found in six structural types of rifampicin solvates and bilayers observed in two types of solvates. Diverse mutual arrangements of these 2D modules lead to the formation of channels with accommodated solvent molecules that consist 10-40% of the crystal volume. DTA/TG analyses and powder X-ray diffraction show that desolvation of the rifampicin 2-propanol solvate results in polymorph II of rifampicin. We show here that solvate


Page 3 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


decomposition may be a route to transformation of a thermodynamically stable polymorphic form into a metastable one.

Introduction Rifampicin is a semisynthetic antibiotic from the group of rifamycins with a broad spectrum of antibacterial activity. It was discovered in 1960’s and it is in use for 40 years. Its pronounced activity against mycobacteria causes that it is still considered as one of the key components of the first-line treatment against tuberculosis. Chemically rifampicin is a complex macrocyclic compound that consists of a chromophore (a naphthohydroquinone system fused with a furanone ring), a mostly aliphatic ansa bridge spanning the chromophore from two opposite sides and the (4-methyl-1-piperazinyl)iminomethyl group attached to the chromophore (Scheme 1). Essential for the activity of rifamycins against bacterial DNA-depended RNA polymerase are four oxygen atoms, two from the chromophore (O1, O8) and two from the ansa bridge (O21, O23) that have to be in a specific spacial arrangement for the effective binding to the enzyme.1-5 The ability of some semi-synthetic rifamycin antibiotics to adopt a zwitterionic structure has also been indicated as a factor increasing their biological activity.6-8 Scheme 1



Page 4 of 38

Rifampicin is chemically rather unstable and according to Biopharmaceutic Classification System it belongs to a class II drugs as it shows high permeability and low solubility.9,10 A search for new crystalline forms of rifampicin that potentially could have improved physicochemical and pharmaceutical properties started early. In 1977 Pelliza et al. reported that this antibiotic can be obtained in two polymorphic forms, I and II, with form II transforming into form I on heating.11 It has been shown that these two forms are monotropically related and form I is thermodynamically more stable.11,12 There have been conflicting reports concerning the molecular structure of rifampicin in the two polymorphic forms. Based in IR spectra it was concluded that both polymorphs consist of the molecules in the phenolic form,11 whereas recent 13C and 15N solid-state NMR and FTIR studies point to the zwitterionic form of rifampicin in polymorph II (Scheme 1).13 Commercially available rifampicin is predominantly pure form II or a mixture of form II either with form I or with amorphous form, although pure form I was also found among the analyzed commercial samples.12,14 In addition to the polymorphic forms, rifampicin easily forms solvates. Pelliza et al. characterized two pentahydrated forms, tetrachloromethane (1/1) solvate and a solvate containing one-ninth of tetrahydrofurane molecule per one rifampicin molecule.11 Later on Henwood et al. added to this list rifampicin mono- and dihydrate, acetone (1/1) and 2-pyrrolidine (1/2) solvates.15 Two hydrated ethylene glycol solvates were obtained serendipitously by de Villiers et al. and they have shown that diethylene glycol cocrystallizes with rifampicin as well.16 Surprisingly, information about crystal structures of solid state forms of rifampicin is very limited. The structure of rifampicin pentahydrate reported at 197517 was for nearly 35 years an important source of information about the molecular structure of this antibiotic, however revision showed that rifampicin molecule exists in these crystals not in the phenolic form as reported, but in the zwitterionic form.18 Currently, the Cambridge Structural Database (CSD, ver. 5.38 with one update)19 contains, in addition to pentahydrate, structural data for two hydrated ethylene glycol solvates,16 dimethanol trihydrate solvate and 1,1,1-trichloroethane (1/1) solvate.6 Rifampicin pentahydrate and dimethanol trihydrate solvate are closely isostructural. Recently, the crystal structure of form I of rifampicin has been determined from X-ray powder diffraction (PXRD) data.20 The structure of ACS Paragon Plus Environment

Page 5 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


rifampicin complexes with several proteins are also known,21-26 including the 3.3 Å resolution crystal structure of the complex of rifampicin with DNA-dependent RNA polymerase.21,24 In the present work crystal structures of thirteen new rifampicin solvates are reported and analyzed.

These solvates were prepared

mostly by recrystallization of rifampicin

from

dichloromethane/aliphatic alcohol mixture, i.e. in conditions preferred by zwitterionic form of rifampicin.6 We have expected that structural information provided by rifampicin solvates might shed light on preferred association mode of zwitterionic form of this antibiotic and, in addition, should allow us to explore changes in molecular structure of this antibiotic as a response to varied intermolecular environments. As desolvation of solvated forms can result in new crystalline forms of a substance, decomposition of rifampicin monohydrate 2-propanol trisolvate was studied by thermal methods and PXRD. Experimental TG-DTA for RIF-2-PrOH was recorded with a SETARAM SETSYS 1200 in open alumina crucible on a sample of 13.8 mg at a heating rate of 5°C min-1 over the temperature range 30–300°C under flow of nitrogen. PXRD was recorded with a Bruker D8 Advance using Cu Kα radiation over a 2θ range of 6-60° with a scan step of 0.050°. Preparation of rifampicin solvates Rifampicin solvates are denoted in this paper as ‘RIF-solvent(x)’ where RIF stands for rifampicin, solvent name is given in the abbreviated form (MeOH, EtOH, 1- or 2-PrOH, 1- or 2-BuOH, 1-, 2- or 3-PenOH, HexOH for methanol, ethanol, propanol, butanol, pentanol and hexanol, respectively, PrO for acetone, BuO for 2-butanone and DMSO for dimethylsufoxide) and x is used when more than one crystal form was obtained from a given solvent. Solvate stoichiometry and presence of water molecules are not indicated in this notation. ACS Paragon Plus Environment


Page 6 of 38

Rifampicin [LOT no. 087K1875] and solvents were purchased from Sigma-Aldrich and used as obtained. Rifampicin (5.5 mg, 0.007 mmol) was placed in an eppendorf tube and dissolved in a mixture of 0.01 ml of dichloromethane and 0.02 ml of another solvent (alcohols: ethanol, 1- and 2-propanol, 1and 2-butanol, 1-, 2- and 3-pentanol, 1-hexanol; ketones: acetone, 2-butanone) and solution was left at room temperature for slow evaporation. When red crystals of solvates started to appear (hours to a few days) eppendorf tubes were tightly closed and kept refrigerated to avoid destruction of crystals by desolvation process. All crystals when removed from the mother liquor were unstable in the air. Solvates RIF-DMSO and RIF-PrO(2) were prepared by

dissolving rifampicin (5.5 mg,

0.007 mmol) in 0.10 ml of DMSO and 0.08 ml of acetone, respectively X-ray crystal structure analyses All diffraction experiments, except that for RIF-EtOH, were carried out at low temperature (120 or 130 K) with an Oxford Diffraction SuperNova diffractometer using mirror-monochromated Cu Kα radiation. Intensity data for RIF-EtOH were measured at 130 K with a KUMA KM4CCD diffractometer using Mo Kα radiation. Diffraction data were processed with the CrysAlis PRO software.27 The structures were solved by direct methods (SHELXS28 or SIR200429) and refined by full-matrix leastsquares on F2 with SHELXL-201430 within the WinGX31 suite of programs. The assumed absolute structure of the studied solvates conforms with the known absolute configuration of rifampicin. In the isostructural series of six monoclinic solvates the unit-cell parameters β were close to 90° what raised some problems with comparison of their crystal structures. To facilitate this process a non-reduced setting with a β angle < 90° was chosen in three cases (RIF-1-BuOH, RIF-2-BuOH and RIF-BuO). Moreover, when the unit cell β angle is close to 90° the crystals are often twinned by pseudomerohedry and this phenomenon was observed for RIF-1-BuOH (domain ratio 55/45) and RIF-BuO (domain ratio 85/15). To highlight a relationship between structures of RIF-PrO(2) and RIF-3-PenOH a non-reduced


Page 7 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


setting was chosen for RIF-PrO(2). The basis vectors (a, b, c) are related to the basis vectors of the reduced setting by (a, b, c) = (a’, b’, c’) [-1 0 0 0 -1 0 1 0 1]. In some crystal structures disorder was observed for rifampicin (RIF-2-BuOH) or solvent (RIF-EtOH, RIF-2-PrOH, RIF-PrO(1), RIF-2-PenOH, RIF-DMSO, RIF-1-PenOH, RIF-1-HexOH) molecules. Whenever necessary restraints were imposed on geometry and displacement parameters of disordered molecules. More details relating to disorder of solvent molecules are included in Supplementary Material. Positions of hydrogen atoms from O-H and N-H groups of rifampicin were determined from electron density difference maps. For phenolic O1-H and amino N40-H groups the O-H and N-H distances were fixed as determined from these maps whereas in the remaining cases the O-H and N-H bond lengths were standardized to 0.84 Å and 0.90 Å, respectively. Positions of the O-H group hydrogen atoms of solvent molecules, whenever possible, were determined from electron density difference maps. In a few cases, positions of hydrogen atoms were deduced on the basis of possible hydrogen bonding interactions. Only in two cases, water molecule O2W in RIF-2-PenOH and O-H group of 2-butanol molecule F in RIF-2-BuOH, the positions of H atoms could not be determined. The C-bound hydrogen atoms of rifampicin and solvent molecules were placed in idealized positions. All hydrogen atoms were refined as riding on their carriers with Uiso(H)=1.2Ueq, except methyl group for which Uiso(H)=1.5Ueq. The summary of crystal structure determinations is given in Table 1.


Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 8 of 38

Table 1. Crystal data and refinement details for rifampicin solvates

solvate type

Type IIa

Type Ia RIF-1-PrOH C43H58N4O12· 3.5(C3H7OH)· (H2O) 1551693

RIF-2-PrOH C43H58N4O12· 3(C3H7OH)· (H2O) 1551685

RIF-1-BuOH C43H58N4O12· 3(C4H9OH)· 0.5(H2O) 1551692

RIF-2-BuOH C43H58N4O12· 3(C4H9OH)· 0.5(H2O) 1551694

RIF-PrO(1) C43H58N4O12· 3.3(C3H6O)· 0.2(H2O) 1551689

RIF-BuO C43H58N4O12· 3(C4H8O)

CCDC no.

RIF-EtOH C43H58N4O12· 2(C2H5OH)· 2.59(H2O) 1551688

formula weight

961.65

1050.78

1021.23

1054.30

1054.80

1018.15

1039.24

crystal system

Orthorhombic

Monoclinic

Monoclinic

Monoclinic

Monoclinic

Monoclinic

Monoclinic

space group

P212121

P21

P21

P21

P21

P21

P21

a (Å)

13.9522 (2)

14.0567 (1)

14.0375 (4)

14.1128 (3)

13.9938 (1)

14.0428 (1)

13.9825 (3)

b (Å)

17.9572 (3)

23.5361 (2)

23.6481 (9)

23.5574 (6)

23.6428 (2)

23.4153 (3)

23.4196 (5)

c (Å)

20.0983 (3)

17.3564 (1)

17.0887 (5)

17.4909 (4)

17.6301 (2)

17.0957 (3)

17.4096 (4)

90

90.275 (1)

91.460 (3)

89.925 (2)

89.695 (1)

90.321 (1)

89.861 (2)

volume (Å )

5035.5 (1)

5742.1 (1)

5670.9 (3)

5815.0 (2)

5832.9 (1)

5621.3 (1)

5701.0 (2)

Z

4

4

4

4

4

4

4

temperature (K)

130

120

120

120

120

130

120

radiation type

Mo Kα

Cu Kα

Cu Kα

Cu Kα

Cu Kα

Cu Kα

Cu Kα

µ (mm-1)

0.10

0.74

0.73

0.72

0.71

0.73

0.72

refl. collected

57414

40237

38727

40903

60673

50275

32444

refl. unique

10232

19640

20447

18852

21720

19140

17586

restrains/parameters

9/647

32/1282

1/1370

72/1267

100/1241

7/1346

49/1264

2

GOF on F

1.100

1.034

1.021

1.045

1.021

1.036

1.027

R1 [I>2σ(I)]

0.0433

0.0622

0.0435

0.0603

0.0748

0.0650

0.0764

wR2 [I>2σ(I)]

0.1124

0.1776

0.1162

0.1609

0.2143

0.1837

0.2137

R1 (all data)

0.0568

0.0625

0.0444

0.0627

0.0753

0.0684

0.0782

0.1294

0.1782

0.1171

0.1623

0.2152

0.1881

0.2174

0.87, -0.52

0.65, -0.46

0.59, -0.44

1.04, -0.68

0.83, -0.75

0.87, -0.55

empirical formula

β (°) 3

wR2 (all data) -3

largest pick and hole (e Å ) 0.41, -0.37


1551690

Page 9 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Table 1. continued solvate type

Type IIIa

Type Ib

Type IVa

Type Va

Type IVb

Type Vb

RIF-DMSO C43H58N4O12· 3(C2H6OS)· 1.73(H2O)

RIF-PrO(2) C43H58N4O12· 3(C3H6O)· (H2O)

RIF-3-PenOH C43H58N4O12· 3(C5H11OH)

RIF-1-PenOH C43H58N4O12· 2.11(C5H11OH) 1.68(H2O)·

RIF-1-HexOH C43H58N4O12· 2.5(C6H14O) (H2O)·

CCDC no.

RIF-2-PenOH C43H58N4O12· 1.88(C5H11OH)· 0.62(CH2Cl2)· 0.5(H2O) 1551683

1551687

1551691

1551686

1551682

1551684

formula weight

1050.32

1088.60

1015.18

1087.37

1039.10

1096.37

crystal system

Monoclinic

Orthorhombic

Monoclinic

Monoclinic

Orthorhombic

Orthorhombic

space group

P21

P212121

P21

P21

C2221

C2221

a (Å)

13.9424 (3)

13.8434 (5)

13.0523 (1)

13.1805 (2)

13.5443 (2)

13.5653 (2)

b (Å)

17.4799 (4)

17.5721 (5)

17.2549 (1)

17.1573 (2)

24.7610 (3)

24.4933 (3)

c (Å)

24.5220 (4)

22.7897 (7)

13.9853 (2)

13.9841 (1)

35.8440 (6)

38.6909 (5)

105.709 (2)

90

118.130 (1)

101.913 (1)

90

90

volume (Å )

5753.1 (2)

5543.8 (3)

2777.7 (1)

3094.3 (1)

12021.0 (3)

12855.4 (3)

Z

4

4

2

2

8

8

temperature (K)

130

130

130

130

130

130

radiation type

Cu Kα

Cu Kα

Cu Kα

Cu Kα

Cu Kα

Cu Kα

µ (mm-1)

1.22

1.81

0.74

0.68

0.69

0.69

refl. collected

18153

35463

14211

17351

18838

19761

refl. unique

14003

10090

7765

10440

9561

10382

restrains/parameters

39/1244

15/674

1/668

1/640

14/647

13/672

GOF on F

1.036

1.022

1.031

1.024

1.031

1.024

R1 [I>2σ(I)]

0.0791

0.0918

0.0269

0.0728

0.0860

0.0842

wR2 [I>2σ(I)]

0.2178

0.2442

0.0731

0.2077

0.2452

0.2476

R1 (all data)

0.0924

0.1022

0.0272

0.0762

0.0892

0.0882

wR2 (all data)

0.2373

0.2551

0.0735

0.2142

0.2504

0.2553

1.16, -1.00

0.18, -0.18

0.86, -0.49

0.85, -0.63

0.84, -0.45

empirical formula

β (°) 3

2

largest pick and hole 0.80, -0.68 (e Å-3)



Page 10 of 38

Results and discussion Rifampicin was recrystallized form a mixture of CH2Cl2 (DCM) and a variety of aliphatic alcohols which were used as received without additional purification or drying. DCM was added to increase solubility of rifampicin and its presence was essential for reproducibility of crystallization experiments that resulted in single crystals of nine new solvates which in eight cases contained, in addition to alcohol molecules, also water molecules. Recrystallization from DCM/3-pentanol mixture resulted in anhydrous RIF-3-PenOH solvate. In one case only, RIF-2-PenOH, DCM was included in crystals alongside alcohol and water. In turn, recrystallization of rifampicin from a DCM/ketone mixture resulted in two solvates, RIF-PrO(1) and RIF-BuO, with the latter solvate being anhydrous. Two other solvates, RIF-DMSO and RIF-PrO(2), were obtained by recrystallization of rifampicin from corresponding solvents without addition of DCM. Thus, two rifampicin solvates were prepared that contained acetone and water molecules however they differed in stoichiometry of their components (Table 1). Classification of rifampicin solvates into different isostructural types, as adopted in this work, is given in Table 1 and will be explained further when discussing the modular character of their structure. X-ray crystallography revealed that

RIF-EtOH is isostructural with the earlier reported

rifampicin solvates: pentahydrate (RIF-H2O; CSD refcode HAXWUA)18 and trihydrate methanol disolvate (RIF-MeOH; CSD refcode MAPHIW).6 We will refer to these three isostructural solvates as of type Ia. In turn, six solvates crystallizing in the monoclinic P21 space group with two rifampicin molecules in the asymmetric unit formed another large isostructural series of crystals (solvates of type IIa). Molecular structure Owing to the presence of acidic (phenols) and basic (amine) groups within the rifampicin molecule, an intramolecular proton transfer can occur resulting in phenolic or zwitterionic (phenolate) forms of this ACS Paragon Plus Environment

Page 11 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


antibiotic (Scheme 1). Earlier studies revealed that rifampicin can crystallize in both forms depending on solvent used for crystallization: neutral rifampicin crystallized from aprotic solvents whereas crystallization from protic solvents, or aprotic solvents with additive of water, resulted in zwitterionic form.6 As expected, in all solvates reported here rifampicin exists in the phenolate form with proton attached to the amine N40 atom of N-methylpiperazine unit. The structure of anhydrous RIF-BuO obtained

from ‘wet’ aprotic 2-butanone solvent proves that even traces of water can shift the

equilibrium in solution from the phenolic towards the phenolate form of rifampicin. In the reported solvates, the proton transfer was confirmed by the location of hydrogen atom at N40 in electron density difference maps, by the analysis of bond lengths involving O8, O11 and N40 and by inspection of hydrogen-bonding interactions of rifampicin. The C8-O8, C11-O11 and C-N40 bond lengths were in the range 1.258(6) – 1.300(7) Å [mean value 1.284(9) Å for 20 data], 1.214(7) – 1.266(4) [mean value 1.250(12) Å for 20 data] and 1.462(12) – 1.514(6) Å [mean value 1.494(10) Å for 60 data] respectively, whereas in the phenolic form found in 1,1,1-trichloroethane solvate (CSD refcode MEPHES) the corresponding geometric parameters are substantially different: the C8-O8 bond length is 1.337(7) Å, C11-O11 1.238(7) Å and the mean C–N40 distance is 1.464(7) Å.6 The observed bond distances C8-O8 and C11-O11 of the phenolate form indicate that a negative charge is strongly delocalized in the chromophore.



Page 12 of 38

Figure 1. Intramolecular hydrogen bonds (dashed lines) in rifampicin molecule with the water molecule O1W bridging O21-H and C15=O15 groups in RIF-PrO(2); only O and N atoms involved in hydrogen bonds are labeled and C-H hydrogen atoms are omitted for clarity. Location of the proton-donor and proton-acceptor groups in zwitterionic rifampicin molecule allows for the formation of five intramolecular hydrogen bonds of varied strength: chain of two O-H···O bonds (O21-H···O23-H···O35=C35) at the ansa bridge and three bonds at the chromophore (O4-H···O11, O1-H···O8, N2-H···N38). Intramolecular hydrogen bond between O23-H and the acetyl group at the ansa chain has been observed in Rifamycin S32 and can also be postulated in polymorph I of rifampicin based on the structure determination from powder diffraction data.20 As this intramolecular interaction is absent in all structures reported here, either of the hydroxyl groups, O21-H or O23-H, might donate proton to the intramolecular O21···O23 hydrogen bond but, nonetheless, O21-H group maintains its proton-donor function in most cases. There are only two exceptions. In RIF-PrO(2), intramolecular O23-H···O21 interaction is an element of three-membered hydrogen-bonded chain where a water molecule acts as a bridge between O21-H and the amide carbonyl group of the same molecule (Fig. 1). In RIF-DMSO the reversed direction of intramolecular hydrogen bond at the ansa chain allows the hydroxyl group O21-H to form intermolecular interaction with the acetyl group of another molecule. Among intramolecular hydrogen bonds at the chromophore, two very strong O-H···O interactions persist in all solvates whereas a weaker N2-H···N38 bond can be broken. This happens when the amide N2-H group is intramolecularly connected through the bridging solvent O-H group to the O21-H group at the ansa chain. In RIF-1-PenOH and RIF-1-HexOH, as well as in the orthorhombic form of rifampicin ethylene glycol solvate (CSD refcode OWELOS),16 these interactions force the amide group to adopt a nearly perpendicular orientation relative to the naphthalene group, thus breaking the intramolecular N2-H···N38 hydrogen bond. Geometry of hydrogen bonds is given in Supporting Information, Table S1.


Page 13 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Superposition of 20 zwitterionic rifampicin molecules from the solvated forms reported in this work (all atoms were used for least-squares superposition); the fragment marked in green shows two different orientations of the amide group; C-H hydrogen atoms are omitted for clarity. Rifampicin has a potential for conformational liability with possible conformational changes occurring mainly within the ansa bridge and also for the substituent at C3 of the chromophore. Conformation of the ansa chain is crucial for antibacterial activity of rifamycin antibiotics as it has to ensure a proper arrangement of the oxygen atoms O1, O8, O21, and O23 that is responsible for correct interaction of rifamycins with the DNA-dependent RNA-polymerase.1-4 Based on conformational analysis of active and inactive forms of rifamycins, Bacchi and co-workers defined the molecular structural parameters associated to the activity of rifamycins.5, 32-33 They indicated that the active pattern with the four oxygen atoms protruding of one molecular face, perpendicularly to the ansa chain and following some defined O···O interatomic distances was the result of the favorable combination of two principal conformational degrees of freedom of the ansa chain.32 It was highlighted that conformational lability of rifamycins in the active form is highly limited, nevertheless the overall arrangement of the pharmacophore is not affected by the rotation at the two junctions between the chromophore and the ansa chain.



Page 14 of 38

Superposition of 20 rifampicin molecules present in the studied solvates is shown in Fig. 2. The interatomic distances between O1, O8 at the chromophore and O21, O23 at the ansa chain [5.678(2) 9.052(2) Å] are for all molecules in the ranges characteristic of active form, confirming thus the limited conformational lability of the ansa chain. The largest conformational differences occur at the junction of the amide group and the chromophore, where the amidic carbonyl group adopts two nearly antiparallel orientations (Fig. 2, marked by a green ellipse). The conformation with the carbonyl group pointing to the side opposite the location of the ansa chain is less populated and was observed in four orthorhombic solvates reported here (RIF-EtOH, RIF-DMSO, RIF-1-PenOH, RIF-1-HexOH). As illustrated in Fig. 2, this conformational change retains the arrangement of pharmacophore practically unaffected.

Figure 3. a) N-Methylpiperazinyl units with the N+-H group pointing to the location side of ansa chain (RIF-1-PrOH) and b) in the opposite direction (RIF-EtOH); c) rifampicin molecule with the chromophore axially attached to piperazine N39 in the orthorhombic form of ethylene glycol solvate;16 C-H hydrogen atoms are omitted for clarity. Another important fragment of the rifampicin molecule that can show some liability is the substituent at C3 of the chromophore. Here two processes can occur: the N-methylpiperazinyl group can rotate about the N38-N39 bond or inversion of configuration at N39 can take place changing the position of the substituent at N39 of piperazine ring from equatorial to axial. The latter situation has been observed in the orthorhombic form of ethylene glycol solvate (Fig. 3c).16 In the solvates reported here, the substituents at the piperazine ring are always situated equatorially, although two different rotamers ACS Paragon Plus Environment

Page 15 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


around the N38-N39 bond are found: those with N40-H group pointing towards the ansa chain and those with the N40-H group oriented in the opposite direction (Fig. 3a,b). We have noticed that orientations of the N40-H group and the amide group are correlated, i.e. the N40-H group is pointing always to the same side of the chromophore as the amidic carbonyl group. Thus, it is highly probable that there is also a correlation between rotations of these two groups within the rifampicin molecule. Assembly of rifampicin molecules via hydrogen bonds and accommodation of solvent molecules As already mentioned, numerous solvated and polymorphic forms of rifampicin have been postulated and reported up today but only a few of them have their crystal structures determined. The crystalline forms with known crystal structure include pentahydrate (RIF-H2O),18 trihydrate methanol disolvate (RIF-MeOH)6 with rifampicin in the phenolate form, two hydrated ethylene glycol solvates with uncertain protonation sites,16 1,1,1,-trichloroethane solvate6 and polymorph I20 of rifampicin where this antibiotic exists in the phenolic form.

Figure 4. (a) Chains via O23-H···O4 hydrogen bonds in solvates of type Ia and (b) narrow channels and small cages accommodating solvent molecules.

RIF-H2O and RIF-MeOH solvates are isostructural, with two water molecules in pentahydrate replaced by two methanol molecules in RIF-MeOH.18 To check whether isostructurality will be retained ACS Paragon Plus Environment


Page 16 of 38

for solvates with other alcohols, rifampicin was recrystallized from solutions containing higher linear homologues of aliphatic alcohols and their branched congeners.

Indeed, recrystallization from

DCM/EtOH mixture resulted in the isostructural form containing two molecules of EtOH and 2.59 molecules of water per one molecule of rifampicin (Table 1). As illustrated in Fig. 4a, in this isostructural series (type Ia solvates) rifampicin molecules are connected via O-H···O hydrogen bonds between the hydroxyl group O23-H from the ansa chain and the phenolic O4-H from the chromophore and form a chain extending along [001] (for geometry of hydrogen bonds see Table S2). There are two water molecules, O1W and O2W, that retain their positions in all three isomorphs (Fig. 5), with the latter molecule acting as a bridge between neighboring chains through hydrogen bonds to the carbonyl oxygen O11 and the phenolate O8 atoms. Water molecule O3W, which in RIF-H2O forms only one hydrogen bond (to the amidic oxygen O15), in alcoholic solvates is replaced by methanol or ethanol molecule.

Figure 5. Location of water molecules in RIF-H2O (green)18 compared with positions of solvent molecules in isostructural RIF-MeOH6 (yellow) and RIF-EtOH (dark violet, this work). ACS Paragon Plus Environment

Page 17 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Further changes are observed in the area of water molecules O4W and O5W which in pentahydrate, together with O1W and O2W, form helical chain via hydrogen bonds. In RIF-MeOH, water molecule O5W is replaced by methanol, whereas in RIF-EtOH this area is disordered and occupied either by one ethanol molecule or one ethanol and one water molecule. In the latter case, the water molecule interacts with two rifampicin molecules in a similar manner to O4W in pentahydrate, but it is not bonded to O2W directly but through the bridging OH group of ethanol.

Figure 6. (a) Chains via hydrogen bonds formed by two symmetry independent molecules in solvates of type IIa and (b) wide channels accommodating solvent molecules

In rifampicin solvates of type Ia, antibiotic molecules and water molecules O1W and O2W form via hydrogen bonds a 3D framework containing small cages and narrow channels (Fig. 4b). Whereas the unit cell volume in RIF-H2O and RIF-MeOH differs only by 79 Å, for RIF-EtOH it increases by 200 Å relative to the methanol solvate. The next alcohol in the homologues series, 1-propanol, is obviously too large to be accommodated in the cages and the narrow channels of this relatively rigid framework. ACS Paragon Plus Environment


Page 18 of 38

When rifampicin was recrystallized from DCM/1-propanol mixture a new rifampicin framework with larger channels was formed (type IIa solvates). Isostructural crystals were obtained when 1-butanol and branched alcohols, 2-propanol and 2-butanol, were used in crystallization in place of 1-propanol. Surprisingly, type IIa solvates were also obtained with aprotic solvents such as acetone or 2-butanone. The degree of hydration of these solvates is significantly lower than in previous series, with 2-butanone solvate being anhydrous. These isostructural solvates crystallize in the monoclinic space group P21 with two symmetry independent molecules of rifampicin, designated as A and B, in the asymmetric unit. The rifampicin molecules are connected into chains propagating along [001] via bifurcated N+-H···O hydrogen bonds formed between piperazine N40-H group and amidic O15 and hydroxyl O1 (Fig. 6a). These 1D chains are further assembled into 2D layers parallel to (100) through O-H···O interactions between the functional groups attached to the ansa chains. Like in solvates of type Ia, solvent molecules are accommodated in channels extending along [100] that, however, are significantly wider (Fig. 6b). Of the six different sites occupied by guest alcohol molecules in this type of solvates, systematically occupied are only two, those of the C and D molecules (Fig. 7a), whereas positions of the remaining molecules, as well as those of water molecules are varying (Fig. S8). In solvates of type IIa obtained from DCM/ketone mixture, positions of four guest molecules C, D, E and F are well retained (Fig. 7b). The fifth molecule, G, is also found at the same site in RIF-PrO(1) and RIF-BuO, however in the latter solvate, owing to disorder, the guest molecule occupies this site only partially.


Page 19 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Common guest sites in solvates of type IIa: a) RIF-1-PrOH (grey), RIF-2-PrOH (red), RIF1-BuOH (dark green), RIF-2-BuOH (blue) and b) RIF-PrO(1) [dark blue] and RIF-BuO (green). H atoms are omitted for clarity.

Recrystallization of rifampicin from DCM/2-pentanol mixture resulted in a new solvate type (type IIIa) that, like solvates of type IIa, crystallized in the monoclinic P21 space group with two rifampicin molecules in the asymmetric unit. In RIF-2-PenOH two symmetry independent rifampicin molecules form separate chains (consisting of molecules A or B) through bifurcated N+-H···O hydrogen bonds (Fig. 8a) whereas in the previous series molecules A and B belonged to the same chain (Fig. 6a). These chains are also further assembled into 2D layers by hydrogen bonds between the functional groups at the ansa chain, however these interactions differ in the two monoclinic forms (Table S2). Nevertheless, solvent molecules are accommodated in the channels extending along [100] (Fig. 8b), that in both crystalline forms consist ca. 28% of the crystal volume whereas in type Ia solvates solvent molecules occupied 10-14% of the crystal space.



Page 20 of 38

Figure 8. (a) Chain via N+-H···O hydrogen bonds between molecules A in rifampicin solvates of type IIIa and (b) channels accommodating solvent molecules

Recrystallization of rifampicin from DCM/3-pentanol mixture, or from acetone, resulted in another series of monoclinic solvates (RIF-3-PenOH - type IVa solvates; RIF-PrO(2) - type IVb solvates), with one rifampicin molecule in the asymmetric unit. Bifurcated N+-H···O hydrogen bonds, analogous to those in type IIa and IIIa solvates, assemble rifampicin molecules into chains propagating along [010] (Fig. 9a) and there are no specific intermolecular interactions between rifampicin molecules from adjacent chains. Solvent molecules are accommodated in a system of intersecting channels extending parallel to (10-1) (Fig. 9b, c). As interactions between chains are weak, the rifampicin framework easily adapts to the requirements of solvent molecules. This is best reflected in the fraction of space occupied by channels in RIF-PrO(2) and RIF-3-PenOH, where it changes from 25.6% to 32.8% respectively.


Page 21 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. (a) Chain via N+-H···O hydrogen bonds formed in rifampicin solvates of type IIIa or IIIb and intersecting channels accommodating solvent molecules in type IIIa solvates (b) and type IIIb solvate (c).

The chains formed via bifurcated N+-H···O hydrogen bonds are typical of the monoclinic solvates of rifampicin and in these crystals the N40+-H vector is always pointing to the side of the ansa chain location. In orthorhombic solvates of type Ia and in RIF-DMSO solvate (type Ib), which is also orthorhombic, the N+-H vector has reversed direction and the N+-H group is hydrogen bonded to one of the DMSO molecules. Specific interactions between rifampicin molecules involve hydroxyl O21-H and acetyl oxygen O35 with O21-H···O35 hydrogen bond assembling molecules into chain propagating along [100] (Fig. 10a). Neighboring chains are bridged by water molecules that donate protons to hydrogen bonds formed with carbonyl O11 and phenolate O8, generating thus a three dimensional



Page 22 of 38

framework of rifampicin and water molecules. Solvent channels within this framework are symmetric with a screw 21 axis directed along their long axis and consist 28.4% of the crystal volume (Fig. 10b).

Figure 10. (a) Chain via O-H···O hydrogen bonds between the groups at the ansa chain in rifampicin solvates of type Ib and (b) wide channels accommodating solvent molecules

Recrystallization of rifampicin from a mixture of DCM and longer linear alcohols, 1-pentanol and 1hexanol, resulted in solvates that are orthorhombic, space group C2221, and crystallize with one molecule in the asymmetric unit. Despite similarity of the unit cell parameters, these two solvates are not isostructural. In RIF-1-PenOH (type Va) rifampicin molecules related by a twofold axis form discrete dimeric assemblies through a pair of N40-H···O15 hydrogen bonds (Fig. 11a) whereas in RIF1-HexOH (type Vb) analogous discrete dimers are further connected via hydrogen bonds between functional groups at the ansa chains (Fig. 11c) into 1D assemblies propagating along [100]. Nevertheless, a system of 3D intersecting channels occupies a similar part of the crystal volume: 38.8% in RIF-1-PenOH and 39.6% in RIF-1-HexOH (Fig. 11b, d).


Page 23 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 11. Hydrogen bonds between rifampicin molecules in solvates of type Va (a) and Vb (c) and intersecting channels accommodating solvent molecules [(b) – type Va, (d) – type Vb]. The rifampicin dimeric motif through N40-H···O15 hydrogen bonds is marked in grey.

Zwitterionic rifampicin preferentially includes into crystals protic solvent molecules (alcohols, water) as was shown by recrystallization of this antibiotic from a mixture of alcohols with DCM. In case of ACS Paragon Plus Environment


Page 24 of 38

aprotic solvents, rifampicin shows much higher selectivity for ketones than for DCM as no DCM was included when crystallization was carried out from a DCM/ketone mixture. In the studied solvates rifampicin molecules form a variety of hydrogen-bonded assemblies (discrete dimers –types Va and Vb; diverse 1D chains – types Ia, Ib, IIa, IIIa, Vb; 2D layers – type IIa, IIIa) that can be extended into higher dimensions by hydrogen bonds to water molecules (type Ia and Ib). Solvent molecules, that occupy 1040% of the crystal volume, are in most cases accommodated in channels, or systems of 2D or 3D intersecting channels, and do not bind extensively to rifampicin via hydrogen bonds.

Periodic rifampicin modules as building blocks of rifampicin solvates It has been noticed that in the studied solvates, except those crystallizing in the C2221 space group, two unit cell parameters have always the values from the ranges 13.9 – 14.1 Å and 17.1-17.9 Å. The analysis of hydrogen bonding motifs did not reveal any recurring structural features that would allow us to explain similarity of these two unit cell parameters. However, a closer look at the crystal structures of solvates allowed us to identify some periodic modules formed from rifampicin that did not result from hydrogen bonding interactions between the antibiotic molecules, but appeared repeatedly in their structures. This gave us the idea to look at the structures of solvates from a point of view of common periodic modules (layers, rods) that do not necessarily correspond with the structural motifs formed via intermolecular hydrogen bonds. The first periodic module that was singled out in all studied solvates of zwitterionic rifampicin was a periodic rod defined by a translation vector of ca. 14.0 Å in length, virtually parallel to the line joining atoms C3 and C12 of the rifampicin molecule (Fig. 12a). A survey of the CSD revealed that analogous 1D periodic modules could also be identified in the crystal structures of other rifamycins that exist in zwitterionic or ionic form (Fig. 12b-d). It should be emphasized that these periodic rods were not found when rifampicin crystallized in the phenolic form (polymorph I and 1,1,1-trichoroethane solvate). Moreover, we have noticed that rifapentine (CSD refcode MAFLAI),34 reported to exist in its crystal in a phenolic form, actually has the phenolate form what is confirmed by molecular geometry and ACS Paragon Plus Environment

Page 25 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hydrogen bonding interactions. These observations suggest that electrostatic interactions between zwitterionic rifamycins may dominate in hierarchy of intermolecular interactions over hydrogen bonding, being an important structure-determining factor.

Figure 12. Two different views of the recurring periodic rods formed by zwitterionic or ionic rifamycins: (a) RIF-1-HexOH (this work); (b) MAPHOC;6 (c) MAFLAI;34 (d) BOBMOU10.35

In rifampicin solvates these periodic rods form a basis for the construction of two types of rifampicin 2D periodic modules: monolayers with the p1211 layer group symmetry (Fig. 13) and bilayers with the c121layer group symmetry (Fig. 14). Some structural modifications of monolayers

resulting, for

example, from different orientation of N-methylpiperazine group relative to the ansa chain may give rise to the formation of N-H···O hydrogen bonds between the molecules within the layer, however conventional hydrogen bonds are not a prerequisite to the formation of these 2D modules.



Page 26 of 38

Figure 13. 2D periodic module characteristic of rifampicin solvates of type Ia, Ib, IIa, IIIa, IIIb, IVa and IVb: a) with bifurcated N+-H···O hydrogen bonds between rifampicin molecules (type IIa, IIIa, IIIb, IVa and IVb solvates); (b) with no conventional hydrogen bond between rifampicin molecules (type Ia and Ib solvates) and (c) its side view. Periodic rods are highlighted in grey.

Figure 14. 2D periodic module characteristic of solvates of type Va and Vb: (a) view of the bilayer along c; (b) a constituent single layer of the bilayer; (c) side view of the bilayer along [1-10] (molecules in single layers shown in black or green).

To systematize structurally solvates of zwitterionic rifampicin, those constructed from the p1211 symmetric monolayers have been classified into seven isostructural types (Ia, Ib, IIa, IIIa, IIIb. IVa, IVb). Solvates with a roman numeral I-III have the same orientation of the adjacent monolayers but differ in their mutual position. For example solvates of type Ia and Ib are both orthorhombic, space group P212121, with similar unit cell parameters. They are not isostructural because in their structures


Page 27 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


monolayers of p1211 symmetry are differently positioned in the direction of their intrinsic twofold screw axis. Such displacement does not influence lattice parameters and crystal symmetry, as schematically illustrated in Fig. 15b, but influences shape and size of the solvent channel and adjusts interlayer hydrogen bonding interactions between rifampicin molecules.

Figure 15. Schematic illustrations of rifampicin solvate structures: (a) two views of a schematic representation of rifampicin molecule: piperazinyl unit - blue, ansa chain - red, chromophore - green. (b) structures of type Ia and Ib: view along a 21 axis of the monolayer (left) and view along the rod direction (right). (c) IIa: view along a pseudo-21 axis of the 2D periodic module (left) and view along the 1D rod direction (right). (d) IIIa and IIIb: view along a 21 axis of the 2D periodic module (left) and view along the 1D rod direction (right). Crystal packing diagrams corresponding to these schematic illustrations are included in Supplementary Material.



In turn, when the structure of type Ib is modified by translational movement

Page 28 of 38

of successive

monolayers in the same direction along a then symmetry of the structure is reduced to P21 (Z’=2) leading to type IIIa and IIIb structures, which just differ in the magnitude of shift along a (Fig. 15d). Solvates of type IIIb are not reported in this work but they correspond to the monoclinic form of ethylene glycole solvate (CSD refcode: OWELUY).1 When the above mentioned movements of successive monolayers occur in the opposite directions along a, the symmetry also changes to P21 (Z’=2), however in this case monolayers become pseudo-symmetric as their twofold screw axes do not coincide with the crystallographic symmetry (Fig. 15b). This kind of monolayer arrangement is found in type IIa solvates, the largest isostructural group of rifampicin solvates. Type IV solvates are also constructed from the p1211 monolayers but in contrast to the solvates of type II and III they cannot be derived from type Ib by simple translational movements of the monolayers. Here adjacent monolayers are related by translation operation perpendicular to the symmetry axis of the layer resulting in the P21 (Z’=1) space group symmetry of the entire crystal. Structures of solvates of type IVa and IVb are related by translational movement of the successive layers in the same direction along a and this is the only movement direction that does not change the crystal symmetry (Fig. 16).


Page 29 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 16. Schematic illustration of the crystal packing of rifampicin monolayers in solvates of type IVa and IVb: view along a 21 symmetry axis of the monolayer (left) and view along the 1D rod (right). For schematic representation of rifampicin see Fig. 15

Rifampicin monolayers described above are constructed from rifampicin periodic rods. As mentioned earlier, these rods also serve for the construction of bilayers (Fig. 14) with the c121 layer group symmetry. These bilayers are found in solvates of type Va and Vb, that crystallize in the C2221 space group. They are parallel to (001) and rods are extending along [110] in one part of the bilayer and along [-110] in the other one. Rifampicin molecules from two different parts of the bilayer are forming C2 symmetric dimers by hydrogen bonds between N40+-H and amidic O15 atom. Solvates of type Va and Vb belong to the same space group, have similar unit cell parameters and the same orientation of bilayers but, analogously to the orthorhombic solvates of type Ia and Ib, differ in position of monolayers along the bilayer intrinsic twofold screw axis. Schematic comparison of these two solvate types is given in Fig. 17.



Page 30 of 38

Figure 17. Schematic illustration of the crystal packing of the c121 bilayers in solvates of type Va and Vb viewed along [110]; (a) type Va ; (b) type Vb.

The above examples illustrate that the crystal structures of rifampicin solvates can be best interpreted by the analysis of crystal packing with the use of two types of recurring 2D periodic modules constructed from identical 1D periodic rods. To our surprise these modules were not defined, as usual, by strong specific interactions such as hydrogen bonds, even though rifampicin molecule has a strong propensity to form such interactions. To identify the forces responsible for the observed organization of zwitterionic rifampicin molecules in the solvated crystalline forms further studies involving computational methods would be necessary. Desolvation of RIF-2-PrOH The PXRD pattern of a rifampicin sample used for crystallization experiments (Fig. 18) was in a good agreement with that of polymorph I of rifampicin (Fig. 18),20 i.e. a thermodynamically stable form of this compound at room temperature where rifampicin exists in the phenolic form. Because in all solvates reported in this work rifampicin adopts a zwitterionic form that was postulated for polymorph II of this antibiotic,13 we have decided to determine the polymorphic form of rifampicin obtained through decomposition of one of its solvates, namely RIF-2-PrOH, representing the most populated type IIa of rifampicin solvates (Fig. 18).


Page 31 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RIF-2-PrOH was subjected to TG-DTA analysis. A gradual weight loss of 16.67% observed by TGDTA methods agrees well with the removal of five 2-propanol and two water molecules before melting (calculated weight loss 16.45%). At the DTA curve two endothermal peaks at 80 and 113 °C, corresponding to the loss of solvent molecules, were followed by a sequence of endo-exo-exo peaks at 190, 205 and 253°C. This fragment of the DTA curve closely resembles the DSC curve reported for polymorph II31 where the endothermal peak at 196°C was attributed to the melting of form II, the exothermal peak at 207°C to crystallization of thermodynamically stable form I and the exothermal peak at 255°C to decomposition of rifampicin. Formation of polymorph II after desolvation process was additionally confirmed by PXRD. A sample of RIF-2-PrOH was kept in an open vial at 120°C for six hours to remove solvent molecules. The recorded PXRD pattern (Fig. 18), revealed formation of polymorph II together with some amount of amorphous form. The recorded PXRD pattern corresponded well with the published pattern of form II.25 The above experiments show that decomposition of rifampicin solvates of type IIa leads to metastable polymorph II of this antibiotic. It also illustrates that in some specific cases solvates may be used to transform thermodynamically stable polymorph into its metastable counterpart. The crystal structure of polymorph II of rifampicin has not been determined yet, however there are some spectroscopic evidences that this polymorph consists of rifampicin molecules in the phenolate form13 consistent with our observations.


what is


Page 32 of 38

Figure 18. Powder diffraction pattern recorded for (a) a rifampicin sample used for preparation of solvates (peaks characteristic of form I are marked in orange) and (c) sample obtained after decomposition of RIF-2-PrOH solvate. b) TG (blue) and DTA (red) patterns recorded for RIF-2-PrOH.

Summary and conclusions Rifampicin crystallizes from polar solvents in solvated forms that in addition to solvent molecules often contain water molecules. In these solvates rifampicin exists as zwitterion. The most conformationally labile parts of the rifampicin molecule are the amidic junction between the ansa chain and the chromophore and the substituent at position 3 of the chromophore. The conformational changes occurring in these parts of the molecule do not affect significantly the arrangement of the four oxygen atoms, important for rifampicin binding to the receptor.

The most striking structural feature of the rifampicin solvates reported in this work is that despite connections via hydrogen bonds between zwitterionic molecules, the repeating structural motif occurring in all solvates is based on nonspecific interactions. The 1D rod with translation vector length ACS Paragon Plus Environment

Page 33 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of ca. 14.0 Å is characteristic not only for rifampicin solvates but can be also identified in crystals of zwitterionic or ionic rifampicin analogues. In rifampicin solvates these rods form a basis for the construction of two types of 2D periodic modules: monolayers of the p1211 layer group symmetry and bilayers of the c121 layer group symmetry. Our analysis of crystal structures shows that the variety of structural forms of rifampicin solvates originates from diverse mutual arrangements of these 2D modules as a response to the shape, size and functionality of the included guest molecules. In addition decomposition of RIF-2-PrOH shows that solvate decomposition may be a route to transformation of a thermodynamically stable polymorphic form into a metastable one.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: rifampicin atom-numbering scheme, description of the disorder, hydrogen-bond tables, figures showing crystal packing of rifampicin layers, arrangement of the solvent molecules in channels (PDF). Accession codes. CCDC 1551682-1551694 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. AUTHOR INFORMATION Corresponding Author



Page 34 of 38

* E-mail: [email protected] * E-mail: [email protected]

ORCID Barbara Wicher: 0000-0003-2254-2508 Maria Gdaniec: 0000-0001-8249-7193

Krystian Pyta: 0000-0001-5250-9990

Piotr Przybylski: 0000-0001-8072-5877

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The research was supported by National Science Centre grants N N204 212940 and 2011/03/B/ST5/01014.

References:


Page 35 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(1). Lancini, G.; Zanichelli, W. Structure-activity relationship in rifamycins. In Structure-activity relationship among the semisynthetic antibiotics; Perlman, D., Ed.; Academic Press: New York, 1977; pp 531-600. (2) Brufani, M.; Cerrini, S.; Fedeli, W.; Vaciago, A. J. Mol. Biol. 1974, 87, 409-435. (3) Brufani, M.; Cellai, L.; Cerrini, S.; Fedeli, W.; Vaciago, A., Mol. Pharmacol. 1978, 14, 693-703. (4) Brufani, M.; Cellai, L.; Cerrini, S.; Fedeli, W.; Segre, A.; Vaciago, Mol. Pharmacol. 1982, 21, 394399. (5) Bacchi, A.; Ginacarlo, P. J. Med. Chem. 1998, 41, 2319-2332. (6) Pyta, K.; Przybylski, P.; Wicher, B.; Gdaniec, M.; Stefanska, J. Org. Biomol. Chem., 2012, 10, 2385-2388. (7) Pyta, K.; Przybylski, P.; Klich, K.; Stefańska, J. Org. Biomol. Chem., 2012, 10, 8223-8297. (8) Bujnowski, K.; Synoradzki, L.; Darłak, R. C.; Zevaco, T. A.; Dinjus, E. RSC Adv., 2016, 6, 114758114772. (9) Singh, S.; Mariappan, T. T.; Sharda, N.; Kumar, S.; Chakraborti, A. K. Pharm. Pharmacol. Comm. 2000, 6, 405-410. (10) Sorokoumova, G. M.; Vostrikov, V. V.; Selishcheva, A. A.; Rogozhkina, E. A.; Kalashnikova, T. Yu.; Shvets, V. I.; Golyshevskaya, V. I.; Martynova, L. P.; Erokhin, V. V. Pharm. Chem. J. 2008, 8, 475-478. (11) Pelizza, G.; Nebuloni, M.; Ferrari, P.; Gallo, G.G. Il Farmaco 1977, 32, 471-481. (12) Agrawal, S.; Ashokraj, Y.; Bharatam, P. V.; Paillai, O.; Panchagnula, R. Eur. J. Pharm., 2004, 22, 127-144. (13) Przybylski, P.; Pyta, K.; Klich, K.; Schilf, W.; Kamieński, B. Magn. Res. Chem. 2014, 52, 10-21. (14) ) Henwood, S.Q.; de Villiers, M. M.; Liebenberg, W.; Lötter, A.P. Drug Dev. Ind. Pharm. 2000, 26, 403-408.



Page 36 of 38

(15) Henwood, S.Q.; Liebenberg, W.; Tiedt, L.R.; Lötter, A.P.; de Villiers, M. M. Drug Dev. Ind. Pharm. 2001, 27, 1017-1029. (16) de Villiers, M. M.; Caira, M. R.; Li, J.; Strydom, S. J.; Bourne, S. A.; Liebenberg, W. Mol. Pharmaceutics, 2011, 8, 877-888. (17) Gadret, M.; Goursolle, M.; Leger, J.M.; Colleter, J. C. Acta Crystallogr. 1975, B31, 1454-1462. (18) Wicher, B.; Pyta, K.; Przybylski, P.; Tykarska, E.; Gdaniec, M. Acta Crystallogr. 2012, C68, o209o212. (19) Groom, C. R.; Bruno, I. J. ; Lightfoot ,M. P.; Ward, S. C. Acta Crystallogr,, 2016, B72, 171-179. (20) Ibiapino, A. L.; Seiceria, R. C.; Pitaluga Jr., A.; Trindade, A. C.; Ferreira, F. F. CrystEngComm, 2014, 16, 8555-8562. (21) Campbell, E. A.; Korzheva, N.; Mustaev, A.; Murakami, K.; Nair, S.; Goldfarb, A.; Darst, S.A. Cell, 2001, 104, 901-912. (22) Chrencik, J. E.; Orans, J. O.; Moore, L. B.; Xue, Y.; Peng, L.; Collins, J. L.; Wisely, G. B.; Lambert, M. H.; Kliewer, S. A.; Redinbo, M. R. Mol. Endocrinol., 2005, 19, 1125-1134 (22) Baysarowich, J.; Koteva, K.; Hughes, D. W.; Ejim, L.; Griffiths, E.; Zhang, K.; Junop, M.; Wright, G. D. Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 4886-4891. (24) Nakashima, R.; Sakurai, K.; Yamasaki, S.; Nishino, K.; Yamaguchi, A. Nature, 2011, 480, 565569. (24) Molodtsov, V.; Nawarathne, I. N.; Scharf, N. T.; Kirchhoff, P. D.; Showalter, H. D.; Garcia, G. A.; Murakami, K. S. J. Med. Chem., 2013, 56, 4758-4763. (25) Liu, L. K.; Abdelwahab, H.; Martin Del Campo, J. S.; Mehra-Chaudhary, R.; Sobrado, P.; Tanner, J. J. J. Biol. Chem., 2016, 291, 21553-21562. (26) Qi, X.; Lin, W.; Ma, M.; Wang, C.; He, Y.; He, N.; Gao, J.; Zhou, H.; Xiao, Y.; Wang, Y.; Zhang, P. Proc. Natl. Acad. Sci. U. S. A., 2016, 113, 3803-3808. ACS Paragon Plus Environment

Page 37 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(27) CrysAlis PRO Software; Agilent Technologies: Yarton, Oxfordshire, England, 2012. (28) Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112-122. (29) Burla, M. C.; Caliandro, R.; Camalli, M.; Carrozzini, B.; Casarano, G. L.; De Caro, L.; Giacovazzo, C., Polidori, G.; Spagna, R. J. Appl. Crystallogr. 2005, 38, 381-388. (30) Sheldrick, G. M. Acta Crystallogr. 2015, C71, 3-8. (31) Farrugia, L. J. J. Appl. Crystallogr. 2012, 45, 849. (32) Bacchi, A.; Carcelli, M.; Ginacarlo, P. New J. Chem., 2008, 32, 1725-1735. (33) Bacchi, A.; Ginacarlo, P. J. Comput. Aided Mater. Des., 1999, 13, 385-396. (34) Zhou, K.; Li, J.; Zheng, D. S. J. Mol. Struct., 2010, 983, 27-31. (35) Arora, S.K. Mol. Pharmacol., 1983, 23, 133-140.



Page 38 of 38

For Table of Contents Use Only

Solvates of zwitterionic rifampicin: recurring packing motifs via nonspecific interactions Barbara Wicher, Krystian Pyta, Piotr Przybylski and Maria Gdaniec

In solvated crystals, zwitterionic rifampicin molecules form recurring 1D motif through nonspecific interactions. These 1D rods are the construction elements of two types of 2D periodic modules: monolayers of the p1211 layer group symmetry and bilayers of the c121 layer group symmetry.


Solvates of Zwitterionic Rifampicin: Recurring ... - ACS Publications

Recommend Documents